Transgenic cover plants containing hemicellulase and cellulase which degrade lignin and cellulose to fermentable sugars

ABSTRACT

The present invention provides transgenic cover crop plants which after harvest degrade the hemicellulose and cellulose therein to fermentable sugars which can further be fermented to ethanol or other products. In particular, the transgenic plants comprise cellulase, hemicellulase and optionally ligninase genes from microbes operably linked to a DNA encoding a signal peptide which targets the fusion polypeptide produced therefrom to an organelle of the plant, in particular, the chloroplasts. When the transgenic plants are harvested, the plants are ground to release the hemicellulase and cellulase which then degrade the hemicellulose and cellulose of the transgenic plants to produce the fermentable sugars.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/999,197, filed Dec. 4, 2007, which is a continuation-in-part of U.S.patent application Ser. No. 11/399,675, filed Apr. 6, 2006, now U.S.Pat. No. 7,371,926, issued May 13, 2008, which is a continuation of Ser.No. 09/981,900 filed Oct. 18, 2001, now U.S. Pat. No. 7,049,485, issuedMay 23, 2006, which claims benefit of 60/242,408, filed Oct. 20, 2000,each of which are incorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

None.

REFERENCE TO A NUCLEOTIDE/AMINO ACID SEQUENCE LISTING APPENDIX SUBMITTEDON A COMPACT DISC

The application contains nucleotide and amino acid sequences which areidentified with SEQ ID NOs. A compact disc is provided which containsthe Sequence Listings for the sequences. The Sequence Listing on thecompact disc is identical to the paper copy of the Sequence Listingprovided with the application.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates to transgenic monocot plants which arecover crops which after harvest degrade cellulose and hemicellulasetherein to fermentable sugars which can further be fermented to ethanolor other products. In particular, the transgenic plants comprisecellulase and hemicellulase and optionally ligninase genes from microbesoperably linked to a DNA encoding a signal peptide which targets thefusion polypeptide produced therefrom to sub-cellular compartments ofthe plant, in particular the chloroplasts, apoplast (cell wall areas),endoplasmic reticulum, mitochondria and vacule. When the cover croptransgenic monocot plants are harvested, the plants are ground torelease the ligninase, cellulase and hemicellulase which then degradethe lignin, cellulose and hemicellulose of the transgenic plants forproduction of fermentable sugars. The ligninase will degrade lignin intoaromatic (non-sugar) compounds to reduce the needs for expensivepretreatment processes and cellulase will convert polysaccharides intofermentable sugars.

(2) Description of Related Art

If human economies are to become more sustainable, then it is imperativethat humans learn how to use the solar energy that is carbon-fixed inplant biomass to meet a larger fraction of energy and raw materialneeds. About 180 billion tons of new plant matter (biomass) is producedannually worldwide. Thus, about 30 tons of plant matter per person isproduced every year. In North America, about three tons of plant matteris used per person every year. That means that the energy value ofnaturally produced biomass is equivalent to ten times the total humanuse of all types of energy. However, because of the difficulty inextracting the energy from plant biomass, most of the energy potentialof the biomass goes unused.

At present, the United States produces ethanol from starch produced incorn grain using amylase enzymes to degrade the starch to fermentablesugars. Much of the ethanol that is produced from corn grain is exportedto Brazil where it is efficiently used to power transportation vehicles.In general, while the corn grain is used in the production of ethanol,the remainder of the corn biomass, i.e., the leaves and stalks, isseldom unused because of the cost in degrading the leaves and stalkscomprising lignins and cellulose, generally in the form oflignocellulose, to fermentable sugars. The lignocellulose in the stalksand leaves of corn biomass represents a tremendous source of untappedenergy that goes unused because of the difficulty and cost of convertingit to fermentable sugars.

Major expenses associated with crop biomass conversion include the costsof pretreatment processes to remove lignin and make the lignocellulosicmatter accessible to cellulase, plus the costs of production ofhydrolysis enzymes such as cellulases in microbial bioreactors.Production of hydrolysis enzymes within the biomass of cereal covercrops especially will reduce or eliminate the needs for the expensiveenzymes produced in microbes. Cereal cover crops are naturally very lowin lignin, meaning that they require very little needs for pretreatmentprocesses. The ligninase produced within these plants will degrade thelignin content of these crops before or during processing. In GreatLakes areas, many acres of cereal cover crops are annually plantedshortly after the crop (soybean, corn, etc) harvest, and then killed inspring by herbicides before planting of the next crop. In Michigan,cereal rye and winter wheat produce 1.7 to 2 tons of dry biomass, whenthey are harvested in late May just before planting soybean. Thesecereal cover crops are planted to reduce soil erosion and nutrientleaching from soil and water sediment, and provide weed suppression. Inaddition, inclusion of these cover crops before planting soybean breakssoybean cyst nematode and white mold cycle and therefore minimizes yieldreductions due to these factors. Considering that 1.8 million acres ofsoybean are planted only in Michigan, and much more in the U.S. andaround the globe, farmers can greatly increase their farm income shouldthey plant these cover crops after harvesting of their corn crop, andthen harvesting and selling them as crop biomass before planting ofsoybean.

Currently, there are four technologies available to convert cellulose tofermentable sugars. These are concentrated acid hydrolysis, dilute acidhydrolysis, biomass gasification and fermentation, and enzymatichydrolysis.

Concentrated acid hydrolysis is based on concentrated acidde-crystallization of cellulose followed by dilute acid hydrolysis tosugars at near theoretical yields. Separation of acid from sugars, acidrecovery, and acid re-concentration are critical unit operations. Theconcentrated sulfuric acid process has been commercialized in the past,particularly in the former Soviet Union, Germany, and Japan. However,these processes were only successful during times of national crisis,when economic competitiveness of ethanol production could be ignored.

Dilute acid hydrolysis occurs in two stages to maximize sugar yieldsfrom the hemicellulose and cellulose fractions of biomass. The firststage is operated under milder conditions to hydrolyze hemicellulose,while the second stage is optimized to hydrolyze the more resistantcellulose fraction. Liquid hydrolyzates are recovered from each stage,neutralized, and fermented to ethanol. As indicated earlier, Germany,Japan, and Russia have operated dilute acid hydrolysis percolationplants off and on over the past 50 years. However, the technologyremains non-competitive for the conversion of cellulose to fermentablesugars for production of ethanol.

In biomass gasification and fermentation, biomass is converted to asynthesis gas, which consists primarily of carbon monoxide, carbondioxide, and hydrogen) via a high temperature gasification process.Anaerobic bacteria are then used to convert the synthesis gas intoethanol.

In early processes embracing enzymatic hydrolysis of biomass to ethanol,the acid hydrolysis step was replaced with an enzyme hydrolysis step.This process scheme was often referred to as separate hydrolysis andfermentation (SHF) (¹Wilke et al., Biotechnol. Bioengin. 6: 155-175(1976)). In SHF, pretreatment of the biomass is required to removedisrupt lignocellulosic matter, remove lignin so making the cellulosemore accessible to the cellulase enzymes. Many pretreatment options havebeen considered, including both thermal and chemical steps. The mostimportant process improvement made for the enzymatic hydrolysis ofbiomass was the introduction of simultaneous saccharification andfermentation (SSF) U.S. Pat. No. 3,990,944 to Gauss et al. and U.S. Pat.No. 3,990,945 to Huff et al.). This process scheme reduced the number ofreactors involved by eliminating the separate hydrolysis reactor and,more importantly, avoiding the problem of product inhibition associatedwith enzymes.

In the presence of glucose, β-glucosidase stops hydrolyzing cellobiose.The build up of cellobiose, in turn, shuts down cellulose degradation.In the SSF process scheme, cellulase enzyme and fermenting microbes arecombined. As sugars are produced by the enzymes, the fermentativeorganisms convert them to ethanol. The SSF process has, more recently,been improved to include the co-fermentation of multiple sugarsubstrates in a process known as simultaneous saccharification andco-fermentation (SSCF).

While cellulase enzymes are already commercially available for a varietyof applications. Most of these applications do not involve extensivehydrolysis of cellulose. For example, the textile industry applicationsfor cellulases require less than 1% hydrolysis. Ethanol production, bycontrast, requires nearly complete hydrolysis. In addition, most of thecommercial applications for cellulase enzymes represent higher valuemarkets than the fuel market. For these reasons, enzymatic hydrolysis ofbiomass to ethanol remains non-competitive.

However, while the above processes have focused on converting celluloseto fermentable sugars or other products, much of the cellulose in plantbiomass is in the form of lignocellulose. Lignin is a complexmacromolecule consisting of aromatic units with several types ofinter-unit linkages. In the plant, the lignin physically protects thecellulose polysaccharides in complexes called lignocellulose. To degradethe cellulose in the lignocellulose complexes, the lignin must first bedegraded. While lignin can be removed in chemi-mechanical processes thatfree the cellulose for subsequent conversion to fermentable sugars, thechemi-mechanical processes are inefficient. Ligninase and cellulaseenzymes, which are produced by various microorganisms, have been used toconvert the lignins and cellulose, respectively, in plant biomass forproduction of fermentable sugars. However, the cost for these enzymes isexpensive. As long as the cost to degrade plant biomass remainsexpensive, the energy locked up in the plant biomass will largely remainunused.

An attractive means for reducing the cost of degrading plant biomass isto make transgenic plants that contain cellulases and hemicellulases.For example, WO 98/11235 to Lebel et al. discloses transgenic plantsthat express cellulases in the chloroplasts of the transgenic plants ortransgenic plants wherein the cellulases are targeted to thechloroplasts. Preferably, the cellulases are operably linked to achemically-inducible promoter to restrict expression of the cellulase toan appropriate time. However, because a substantial portion of thecellulose in plants is in the form of lignocellulose, extracts from thetransgenic plants are inefficient at degrading the cellulose in thelignocellulose.

U.S. Pat. No. 5,981,835 to Austin-Phillips et al. discloses transgenictobacco and alfalfa which express the cellulases E2, or E3 fromThermomononospora furca. The genes encoding the E2 or E3, which weremodified to remove their leader sequence, were placed under the controlof a constitutive promoter and stably integrated into the plant genome.Because the leader sequence had been removed, the E2 or E3 productpreferentially accumulated in the cytoplasm of the transgenic plants.However, because the cellulase can interfere with metabolic activitiesin plant cytoplasm, the growth of the transgenic plants can be impaired.

U.S. Pat. No. 6,013,860 to Himmel et al. discloses transgenic plantswhich express the cellulase E1 from Acidothermus cellulolyticus. Thegene encoding E1, which was modified to remove the leader region, wasplaced under the control of a plastid specific promoter and the enzymespreferably produced in plastid.

While the above transgenic plants are an improvement, accumulation ofcellulytic enzymes in the cytoplasm of a plant is undesirable sincethere is the risk that the cellulase will interact with metabolicactivities in cytoplasm, and injure the plant. For example, research hasshown that plants such as the avocado, bean, pepper, peach, poplar, andorange also contain cellulase genes, which are activated by ethyleneduring ripening and leaf and fruit abscission. Therefore, transgenicplants which contain large quantities of cellulase in the cytoplasm areparticularly prone to damage. Furthermore, the cellulases accumulate inall tissues of the plant which can be undesirable. Restriction ofcellulase expression to plastids and/or other sub-cellular compartmentsis desirable because it reduces the risk of plant damage due theaccumulation of cellulases in the cytoplasm of the cells. However, formost crop plants, it has been difficult to develop a satisfactory methodfor introducing heterologous genes into the genome of plastids.Furthermore, cellulase is expressed in all tissues which containplastids which can be undesirable.

For production of ligninases to use in degrading lignins, the ligninasesof choice are from the white-rot fungus Phanerochaete chrysosporium. Oneof the major lignin-degrading, extracellular enzymes produced by P.chrysosporium is lignin peroxidase (LIP). Potential applications of LIPinclude not only lignin degradation but also biopulping of wood andbiodegradation of toxic environmental pollutants. To produce largequantities of LIP, the fungus can be grown in large reactors and theenzyme isolated from the extracellular fluids. However, the yields havebeen low and the process has not been cost-effective. Production ofrecombinant LIP in E. coli, in the fungus Trichoderma reesei, andbaculovirus have been largely unsuccessful. Heterologous expression oflignin-degrading manganese peroxidase in alfalfa plants has beenreported; however, the transgenic plants had reduced growth andexpression of the enzyme was poor (²Austin et al., Euphytica 85: 381-393(1995)).

Although difficult to sufficiently and cheaply produce ligninases innon-plant systems, ligninases have evoked worldwide interest because oftheir potential in degrading a variety of toxic xenobiotic compoundssuch as PCBs and benzo(a)pyrenes in the environment (³Yadav et al.,Appl. Environ. Microbiol. 61: 2560-2565 (1995); ⁴Reddy, Curr. Opin.Biotechnol. 6: 320-328 (1995); ⁵Yadav at al., Appl. Environ. Microbiol.61: 677-680 (1994)).

Therefore, a need remains for an economical method for making transgeniccrop plants wherein the ligninase and cellulase genes are incorporatedinto the plant genome but wherein the ligninase and cellulase expressionare restricted to particular plant tissues, e.g., the leaves, and theligninase and cellulase products are directed to a plant organellewherein it accumulates without damaging the transgenic plant.

SUMMARY OF THE INVENTION

Cereal cover crops such as oat, barley, early and late winter wheat,triticale and rye are used to extend the season of active nutrientuptake and living soil cover. Therefore, they reduce nutrient losses inwater and sediment. They are also used to reduce soil erosion, reducenitrogen leaching, and provide weed and pest suppression and increasesoil organic matter.

Winter cereal cover crops are planted shortly before or soon after thecash crop harvest. Then these cover crops are killed before or soonafter planting of the next cash crops in spring. Cereal grains such sthose listed above, are excellent cover crops because they grow rapidlyin cool weather, withstanding moderate frost, and their seed isrelatively inexpensive. The total costs for production of cereal covercrops, including seeding, purchasing seeds, fertilizer and herbicidewithout tillage is only about $40-$50 per acre.

The present invention provides a method for converting lignocellulose ofplant material to fermentable sugars comprising: (a) providing atransgenic monocot plant includes at least one DNA encoding a cellulaseand hemicellulase which is operably linked to a nucleotide sequenceencoding a signal peptide wherein the signal peptide directs thecellulase to an organelle of the transgenic plant and optionally atleast one DNA encoding a ligninase which is operably linked to anucleotide sequence encoding a signal peptide wherein the signal peptidedirects the ligninase to the organelle of the transgenic plant; (b)growing the transgenic plant for a time sufficient for the transgenicplant to accumulate a sufficient amount of the cellulase andhemicellulase and the ligninase if present in the sub-cellularcompartments of the transgenic plant; (c) harvesting the transgenicplant which has accumulated the cellulase, hemicellulase and ligninaseif present in the sub-cellular compartments of the transgenic plant; (d)grinding the transgenic plant for a time sufficient to produce the plantmaterial wherein the cellulase, hemicellulase and ligninase if presentproduced by the transgenic plant are released from organelle and cellwall areas of the transgenic plant; (e) incubating the plant materialfor a time sufficient for the cellulase, hemicellulase and ligninase ifpresent in the plant material to produce the fermentable sugars andlignin deconstructed by the ligninase from the lignocellulose in theplant material; and (f) extracting the fermentable sugars produced fromthe cellulose and hemicelluloses by the cellulase and hemicellulase fromthe plant material.

The present invention further provides a method for convertinglignocellulose of plant material from a cover crop with low amounts oflignin to fermentable sugars comprising: providing a transgenic monocotplant which is the cover crop which includes at least one DNA encoding acellulase and hemicellulase which is operably linked to a nucleotidesequence encoding a signal peptide wherein the signal peptide directsthe cellulase to an organelle of the transgenic plant and optionally atleast one DNA encoding a ligninase which is operably linked to anucleotide sequence encoding a signal peptide wherein the signal peptidedirects the ligninase to the organelle of the transgenic plant; growingthe transgenic plant for a time sufficient for the transgenic plant toaccumulate a sufficient amount of the cellulase and hemicellulase andlimited amounts of the ligninase if present in the sub-cellularcompartments of the transgenic plant; harvesting the transgenic plantwhich has accumulated the cellulase, hemicellulase and ligninase ifpresent in the sub-cellular compartments of the transgenic plant;grinding the transgenic plant for a time sufficient to produce the plantmaterial wherein the cellulase, hemicellulase and ligninase if presentproduced by the transgenic plant are released from the organelle andcell wall areas of the transgenic plant; incubating the plant materialfor a time sufficient for the cellulase, hemicellulase and ligninase ifpresent in the plant material to produce the fermentable sugars andlignin deconstructed by the ligninase from the lignocellulose in theplant material; and extracting the fermentable sugars produced from thecellulose and hemicelluloses by the cellulase and hemicellulase from theplant material. In further embodiments, the DNA encoding the cellulaseis from an organism selected from the group consisting of Trichodermareesei, Acidothermus ceilulyticus, Streptococcus salivarius, Actinomycesnaeslundi, and Thermomonospora fusca. In further still embodiments, theDNA encoding the cellulase is selected from the group consisting of ane1 gene from Acidothermus cellulyticus, a cbh1 gene from Trichodermareesei, a dextranase gene from Streptococcus salivarius, and abeta-glucosidase gene from Actinomyces naeslundi. In still furtherembodiments, the e1 gene comprises the nucleotide sequence set forth inSEQ ID NO:4, the cbh1 gene comprises the nucleotide sequence set forthin SEQ ID NO:10, the dextranase gene comprises the nucleotide sequenceset forth in SEQ ID NO:8, and the beta-glucosidase gene comprises thenucleotide sequence set forth in SEQ ID NO:6. In still furtherembodiments, the DNA encoding the ligninase is from Phanerochaetechrysosporium. In further still embodiments, the hemicellulase orxylanase is Xyl1 from Cochliobolus carbonum. In further embodiments, theligninase is ckg4 comprising the nucleotide sequence set forth in SEQ IDNO:11 or ckg5 comprising the nucleotide sequence set forth in SEQ IDNO:13. In still further embodiments, DNA encoding the cellulase and theDNA encoding the ligninase are each operably linked to a leaf-specificpromoter. In further still embodiments, the leaf-specific promoter is apromoter for rbcS. In still further embodiments, the nucleotide sequenceencoding the signal peptide encodes a signal peptide of rbcS. In furtherstill embodiments, the rbcS comprises the nucleotide sequence set forthin SEQ ID NO:1. In a further still embodiment for any one of theaforementioned transgenic plants of the present invention, thetransgenic plant is selected from the group consisting of rice, wheat,barley and rye. In still further embodiments, the first and second DNAsare stably integrated into nuclear or plastid DNA of the transgenicplant. In further still embodiments, the transgenic plants furtherinclude a DNA encoding a selectable marker operably linked to aconstitutive promoter. In still further embodiments, the DNA encodingthe selectable marker provides the transgenic plant with resistance toan antibiotic, an herbicide, or to environmental stress. In furtherstill embodiments, the DNA encoding resistance to the herbicide is a DNAencoding phosphinothricin acetyl transferase which confers resistance tothe herbicide phosphinothricin. In still further embodiments, thesub-cellular compartment of the transgenic plant is selected from thegroup consisting of nucleus, microbody, endoplasmic reticulum, endosome,vacuole, mitochondria, chloroplast, or plastid. In further stillembodiments, the sub-cellular compartments of the transgenic plant arethe chloroplast, apoplast (cell wall areas), endoplasmic reticulum,mitochondria or vacuole. In still further embodiments, the plantmaterial further includes a plant material made from a non-transgenicplant. In further still embodiments, the hemicellulase is fromCochliobolus carbonum. In still further embodiments, the hemicellulaseis Xyl1 gene encoding a xylanase.

OBJECTS

It is an object of the present invention to provide cover croptransgenic plants which degrade lignin of lignocellulose and deconstructcellulose and hemicellulase to hexos (6 carbon) and pentose (5 carbon)fermentable sugars, methods for making the transgenic plants whichdegrade lignocellulose, and methods for using the transgenic plants todegrade cellulose and hemicellulose to fermentable sugars.

These and other objects of the present invention will becomeincreasingly apparent with reference to the following drawings andpreferred embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 is a diagram of a plasmid containing a heterologous geneexpression cassette containing cbh1 operably linked to the rbcS promoterand DNA encoding the rbcS signal peptide and a heterologous geneexpression cassette containing the bar gene operably linked to the Act1promoter. rbcSP is the rbcS gene promoter, SP is DNA encoding the rbcSsignal peptide, pin3′ is the 3′ untranslated region of the potatoinhibitor II-chloramphenicol acetyltransferase gene, Act1 is thepromoter for the act1 gene, and nos is the 3′ untranslated region of theAgrobacterium nopaline synthase gene.

FIG. 2 is a diagram of a plasmid containing a heterologous geneexpression cassette containing e1 operably linked to the rbcS promoterand DNA encoding the rbcS signal peptide and a heterologous geneexpression cassette containing the bar gene operably linked to the Act1promoter. The terms in the diagram are as in FIG. 1.

FIG. 3 is a diagram of a heterologous gene expression cassettecontaining the bar gene in plasmid pDM302. Act1 is the promoter for theact1 gene and nos is the 3′ untranslated region of the Agrobacteriumnopaline synthase gene.

FIG. 4 is a diagram of plasmid pSMF13 which is plasmid pSK containing aheterologous gene expression cassette containing cbh1 operably linked tothe rbcS promoter. The terms in the diagram are as in FIG. 1.

FIG. 5 is a diagram of plasmid pSMF14 which is plasmid pSK containing aheterologous gene expression cassette containing cbh1 operably linked tothe rbcS promoter and DNA encoding the rbcS signal peptide. The terms inthe diagram are as in FIG. 1.

FIG. 6 is a diagram of plasmid pSMF15 which is plasmid pBI221 containinga heterologous gene expression cassette containing syn-cbh1 operablylinked to the rbcS promoter and DNA encoding the rbcS signal peptide.The terms in the diagram are as in FIG. 1.

FIG. 7 is a diagram of plasmid pTZA8 which is plasmid pBI121 containinga heterologous gene expression cassette containing e1 operably linked tothe CaMV35S promoter and DNA encoding the SSU signal peptide. SSU is theglycine max (soybean) rbcS signal peptide. CaMV35S is the cauliflowermosaic virus 35S promoter. The remainder of the terms are as in thediagram are as in FIG. 1.

FIG. 8 is a diagram of plasmid pZA9 which is plasmid pBI121 containing aheterologous gene expression cassette containing e1 operably linked tothe CaMV35S promoter and DNA encoding the VSP signal peptide. VSP is thesoybean vegetative storage protein beta-leader sequences. The remainderof the terms in the diagram are as in FIG. 7.

FIG. 9 is a diagram of plasmid pZA10 which is plasmid pBI121 containinga heterologous gene expression cassette containing e1 operably linked tothe CaMV35S promoter. The remainder of the terms in the diagram are asin FIG. 7.

FIG. 10 is a diagram of a plasmid containing a heterologous geneexpression cassette containing ckg4 operably linked to the rbcS promoterand DNA encoding the rbcS signal peptide and a gene expression cassettecontaining the bar gene operably linked to the Act1 promoter. Theremainder of the terms in the diagram are as in FIG. 1.

FIG. 11 is a diagram of a plasmid containing a heterologous geneexpression cassette containing ckg5 operably linked to the rbcS promoterand DNA encoding the rbcS signal peptide and a gene expression cassettecontaining the bar gene operably linked to the Act1 promoter. Theremainder of the terms in the diagram are as in FIG. 1.

FIG. 12 is a diagram of plasmid pSMF18 containing a heterologous geneexpression cassette containing ckg4 operably linked to the rbcSpromoter. The remainder of the terms in the diagram are as in FIG. 1.

FIG. 13 is a diagram of plasmid pSMF19 containing a heterologous geneexpression cassette containing ckg5 operably linked to the rbcSpromoter. The remainder of the terms in the diagram are as in FIG. 1.

FIG. 14 is a diagram of plasmid pSMF16 containing a heterologous geneexpression cassette containing ckg4 operably linked to the rbcS promoterand DNA encoding the rbcS signal peptide. The remainder of the terms inthe diagram are as in FIG. 1.

FIG. 15 is a diagram of plasmid pSMF17 containing a heterologous geneexpression cassette containing ckg5 operably linked to the rbcS promoterand DNA encoding the rbcS signal peptide. The remainder of the terms inthe diagram are as in FIG. 1.

FIG. 16 is a diagram of a construct containing the Cochliobolus carbonumendoxylanase/hemicellulase cDNA regulated by the 35S promoter andenhancer. This construct contains the sequences encoding the tobaccopathogenesis-related protein 1a (Pr1a) signal peptide for targeting ofXYL1 into plant apoplast.

The abbreviations are:

CaMV 35S: Cauliflower Mosaic Virus 35S Promoter

Ω: Tobacco Mosaic Virus translational enhancerPr1a SP: the sequence encoding the tobacco

FIG. 17 is a diagram of a construct containing the bar herbicideresistance selectable marker gene controlled by rice actin 1 promoterand Nos terminator.

The abbreviations are:Act1-5′: Rice acting 1 promoterHva1: Barley Leah Protein coding sequencesPinII-3′: Potato proteinase inhibitor terminator

FIGS. 18A, 18B and 18C are diagrams showing commercially availablecassettes.

FIG. 19 is a diagram showing the insertion of genes (gene B) encoding acellulose, hemicellulase, or ligninase into a sequence of DNA, fortargeting to a sub-cellular compartment of the plant.

FIGS. 20 to 24 are diagrams of various gene constructs used for genetictransformation of E1, CBH1, xylanase or ligninase genes targeted intodifferent sub-cellulase compartments of cover crops.

The abbreviations are:RbcS1-P: Chrysanthemum Ribulose bisphosphate carboxylase (Rubisco)promoter

E1: Endoglucanase CBH1: Exoglucanase CKG4: Ligninase

RbcS1-T: Chrysanthemum Ribulose bisphosphate carboxylase (Rubisco)terminatorSPS: signal peptide for secretion derived from sea anemone equistatinSPC: chloroplast signal peptide from Chrysanthemum morifolium smallsubunit proteinSPM: yeast CoxIV secretion (mitochondrial) signal

DESCRIPTION OF THE PREFERRED EMBODIMENTS

All patents, patent applications, government publications, governmentregulations, and literature references cited in this specification arehereby incorporated herein by reference in their entirety. In case ofconflict, the present description, including definitions, will control.

The term “cellulase” is used herein as a generic term that includesendoglucanases such as the E1 beta-1,4-endoglucanase precursor gene (e1)of Acidothermus cellulolyticus and exoglucanases such as thecellobiohydrolase gene (cbh1) of Trichoderma reesei (also classified bysome as Trichoderma longibrachiatum), the dextranase gene ofStreptococcus salivarius encoding the 1,6-alpha-glucanhydrolase gene,and the beta-glucosidase gene from Actinomyces naeslundi. Endoglucanasesrandomly cleave cellulose chains into smaller units. Exoglucanasesinclude cellobiohydrolases, which liberate glucose dimers (cellobiose)from the ends of cellulose chains; glucanhydrolases, which liberateglucose monomers from the ends of cellulose chains; and,beta-glucosidases, which liberate D-glucose from cellobiose dimers andsoluble cellodextrins. When all of the above enzymes are combined, theywork synergistically to rapidly decrystallize and hydrolyze cellulose tohexose fermentable sugars. Similarly, hemicellulases will deconstructthe hemicellulases (endo-xylanase, exoxylase) into pentose fermentablesugars.

The term “lignin” is used herein as a generic term that includes bothlignins and lignocelluloses.

The term “ligninase” is used herein as a generic term that includes allvarieties of enzymes which degrade lignins such as the lignin peroxidasegene of Phanerochaete chrysosporium.

A variety of fungi and bacteria produce ligninase and cellulase enzymes,and based on evolutionary pressures, these fungi are able to degradelignin or cellulose and hemicellulose of plant residues in the soil. Inthe laboratory, cellulases have been used to hydrolyze or convertcellulose and hemicellulose into mixtures of simple sugars that can beused in fermentation to produce a wide variety of useful chemical andfuel products, including but not limited to, ethanol, lactic acid, and1,3-propanediol, which is an important molecular building block in theproduction of environmentally-friendly plastics.

The biodegradation of lignin on cereals that are not cover crops, whichcomprises 20-30% of the dry mass of woody plants, is of great economicimportance because these pretreatment processes are at costs of $1.20 to$2.20 per gallon of ethanol and therefore an important rate-limitingstep in production of alcohol fuels. Furthermore, there is considerablepotential for the transformation of lignin into aromatic chemicalfeedstock. Also, delignification of lignocellulosic feeds has been shownto increase their digestibility by cattle by about 30%, therefore,contributing to enhanced cost effectiveness for producing milk and meat.Moreover, research on lignin biodegradation has important implicationsin biopulping and biobleaching in the paper industry.

The present invention provides transgenic monocot cover crop plantswhich produce cellulases, and optionally ligninases, in the leaves andstraw/stalks of the plant. While the transgenic plant can be any plantwhich is practical for commercial production, it is preferable that thetransgenic plants be constructed from plants which are produced in largequantities and which after processing produce a substantial amount ofleaves and stalks as a byproduct. Therefore, it is desirable that thecover crop transgenic plant be constructed from plants including, butnot limited to rice, wheat, barley and rye.

Thus, the transgenic cover crop plants of the present invention providea plentiful, inexpensive source of fungal or bacterial cellulases,hemicellulases, which can be used to degrade cellulose and hemicellulosein plants to fermentable sugars for production of ethanol or for otheruses. Also fungal and bacterial ligninases that convert lignin residuesinto aromatic compounds such as pre-treating silage to increase theenergy value of lignocellulosic feeds for cows and other ruminantanimals, pre-treating lignocellulosic biomass for fermentativeconversion to fuels and industrial chemicals, and biodegradation ofchloroaromatic environmental pollutants. Because the transgenic plantsof the present invention produce the ligninases, cellulases, or boththerein, the external addition of ligninases and cellulases fordegradation of the plant material is no longer necessary. Therefore, thepresent invention enables the plant biomass, which is destined to becomeethanol or other products, to serve as the source of ligninase andcellulase. Furthermore, the plant material from the transgenic plants ofthe present invention can be mixed with non-transgenic plant material.The cellulases and ligninases, or both produced by the transgenic plantswill degrade the lignin and cellulose of all the plant material,including the non-transgenic plant material. Thus, ligninase andcellulase degradation of plant material can be carried out moreeconomically.

The transgenic plants of the present invention comprise one or moreheterologous gene expression cassettes containing DNA encoding at leastone fungal or bacterial ligninase, cellulase, or both inserted into theplant's nuclear genome. The preferred cellulase is encoded by a DNA fromthe microorganism Acidothermus cellulolyticus, Thermomonospora fusca,and Trichoderma reesei (Trichoderma longibrachiatum). Othermicroorganisms which produce cellulases suitable for the presentinvention include Zymomonas mobilis, Acidothermus cellulolyticus,Cloostridium thermocellum, Eiwinia chrysanthemi, Xanthomonas campestris,Alkalophilic Baccilus sp., Ceilulomonas fimi, wheat straw mushroom(Agaricus bisporus), Ruminococcus flavefaciens, Ruminococcus albus,Fibrobacter succinogenes, and Butyrivibrio fibrisolvens. Also thetransgenic plants of the present invention comprise of cellulases,hemicellulases and ligninases that exist in guts of carpenter ant,termite, and in microbes that exist in the guts of rumen animals.

The preferred ligninase is lignin peroxidase (LIP) encoded in DNA fromPhanerochaete chrysosporium or Phlebia radiata. One of the majorlignin-degrading, extracellular enzymes produced by P. chrysosporium isLIP. The LIPs are glycosylated heme proteins (MW 38 to 46 kDa) which aredependent on hydrogen peroxide for activity and catalyze the oxidativecleavage of lignin polymer. At least six heme proteins (H1, H2, H6, H7,H8, and H10) with LIP activity have been identified in P. chrysosporiumstrain BKMF-1767 of which isozymes H2, H6, H8, and H10 are the majorLIPs in both static and agitated cultures of P. chrysosporium. However,other fungi which produce ligninases suitable for use in the presentinvention include Bjerkandera adusta, Trametes hirsuta, Plebia radiata,Pleurotus spp., Stropharia aurantiaca, Hypholoma fasciculare, Trametesversicolor, Gymnopilus penetrnas, Stereum hirsutum, Mycena haematopus,and Armillaria mellea.

In the present invention, the transgenic plant comprises a DNA encodingone or more cellulase fusion proteins wherein the DNA encoding thecellulases are operably linked to a DNA encoding a signal peptide whichdirects the cellulase fusion protein to a plant organelle such as thenucleus, a microbody (e.g., a peroxisome, or specialized versionthereof, such as a glyoxysome), an endoplasmic reticulum, an endosome, avacuole, a mitochondria, a chloroplast, or a plastid. By sequesteringthe cellulase fusion proteins in the plant organelle, the cellulasefusion protein is targeted for storage into sub-cellular compartmentsincluding chloroplast, apoplast, endoplasmic reticulum, mitochondria,vacuole to avoid their interactions with cytoplasmic activities andharming of transgenic plants. In particular embodiments of the presentinvention, the gene encoding the cellulase is modified by replacing theamino acid codons that encode the leader region of the cellulase withamino acid codons that encode the signal peptide.

In a preferred embodiment of the invention, the amino acid codons thatencode the signal peptide that directs the protein to which it isattached to the plant organelle, the chloroplasts, are the nucleotidecodons that encode the rice rubisco synthase gene (rbcS) small subunitsignal peptide (rbcSSP). The nucleotide sequence of the rbcS is setforth in SEQ ID NO:1 (GenBank Accession No. X07515). The 47 amino acidsignal peptide of the rbcS protein has the amino acid sequence MAPPSVMASS ATIVA PFQGS SPPPA CRRPP SELQL RQRQH GGRIR CM (SEQ ID NO:2). TherbcS SP directs proteins to which it is operably linked to thechloroplasts of the transgenic plant. Therefore, in the preferredembodiment of the present invention, the transgenic plant comprises aDNA encoding the cellulase operably linked with a DNA encoding the rbcSSP to produce the cellulase fusion protein. The rbcS SP directs thecellulase fusion protein to the chloroplasts. Thus, the cellulase fusionprotein produced by the transgenic plant accumulates in the chloroplastsof the transgenic plant which protects the transgenic plant fromdegradation by the cellulase fusion protein while it is beingcultivated. Alternatively, the DNA encoding the cellulase is modified atits 3′ end to encode a transit peptide such as the peptide RAVARL (SEQID NO:3), which targets the ligninase fusion protein to the peroxisomes(U.S. Pat. No. 6,103,956 to Srienc et al.). Preferably, the leaderregion of the cellulase is also removed. In any one of the aboveembodiments, the cellulase can be further modified to include a GCcontent that approximates the GC content of the genomic DNA of the plantby methods well known in the art.

In a preferred embodiment, the cellulase comprising the cellulase fusionprotein is encoded by the E1 beta-1,4-endoglucanase precursor gene (e1)of Acidothermus cellulolyticus, the cellobiohydrolase gene (cbh1) ofTrichoderma reesei (Trichoderma longibrachiatum), the beta-glucosidasegene from Actinomyces naeslundi, or the glucanhydrolase (dextranase)gene from Streptococcus salivarius. The nucleotide sequence of the e1DNA is set forth in SEQ ID NO:4 (GenBank Accession No. U33212), whichencodes the cellulase with the amino acid sequence set forth in SEQ IDNO:5. SEQ ID NO:6 provides the nucleotide sequence of thebeta-glucosidase gene from Actinomyces naeslundi (GenBank Accession No.AY029505), which encodes the beta-glucosidase with the amino acidsequence set forth in SEQ ID NO:7. SEQ ID NO:8 provides the nucleotidesequence of the dextranase gene from Streptococcus salivarius (GenBankAccession No. D29644), which encodes a glucanhydrolase with the aminoacid sequence set forth in SEQ ID NO:9. The nucleotide sequence of cbh1is set forth in SEQ ID NO:10 (GenBank Accession No. E00389), whichencodes the cellulase that includes the joined exons from positions 210to 261, 738 to 1434, and 1498-1881.

In the present invention, the transgenic plant comprises a DNA encodingone or more ligninase fusion proteins wherein a DNA encoding theligninase is operably linked to a DNA encoding a signal peptide whichdirects the ligninase fusion protein to a plant organelle. Bysequestering the ligninase fusion proteins in the plant sub-cellularcompartments, the modified ligninase is prevented from damaging thecytoplasm of transgenic plants while the plant is being cultivated. Inparticular embodiments of the present invention, the leader sequence ofthe gene encoding the ligninase is modified by replacing the amino acidcodons that encode the leader region of the ligninase with amino acidcodons that encode the signal peptide.

In a preferred embodiment of the invention, the amino acid codons thatencode the signal peptide are the amino acid codons which encode therice rubisco synthase gene (rbcS) small subunit signal peptide (rbcSSP).The nucleotide sequence of the rbcS is set forth in SEQ ID NO:1 (GenBankAccession No. X07515). The 47 amino acid signal peptide of the rbcSprotein has the amino acid sequence MAPPS VMASS ATIVA PFQGS SPPPA CRRPPSELQL RQRQH GGRIR CM (SEQ ID NO:2). Therefore, in the preferredembodiment of the present invention, the transgenic plant comprises aDNA encoding the ligninase operably linked to a DNA encoding the rbcSSP. The rbcS SP directs the ligninase fusion protein to thechloroplasts. Thus, the ligninase fusion protein produced by thetransgenic plant accumulates in the chloroplasts of the transgenic plantwhich protects the transgenic plant from degradation by the ligninasefusion protein while it is being cultivated. Alternatively, the DNAencoding the ligninase is modified at its 3′ end to encode a transitpeptide such as the peptide RAVARL (SEQ ID NO:3). Optionally, the leaderregion of the ligninase is also removed. In any one of the aboveembodiments, the ligninase can be further modified to include a GCcontent that approximates the GC content of the genomic DNA of the plantby methods well known in the art.

In a preferred embodiment of the invention, the ligninase comprising theligninase fusion protein is encoded by the lignin peroxidase gene (LIP)genes ckg4 (H2) and ckg5 (H10) of Phanerochaete crysosporium (³de Boeret al., Gene 6: 93-102 (1987), ⁷Corrigendum in Gene 69: 369 (1988)). Thenucleotide sequence of the ckg4 gene is set forth in SEQ ID NO:11(GenBank Accession No. M18743), which encodes the amino acid with thesequence set forth in SEQ ID NO:12. The nucleotide sequence of the ckg5gene is set forth in SEQ ID NO:13 (GenBank Accession No. M18794), whichencodes the amino acid with the sequence set forth in SEQ ID NO:14.

In the present invention, transcription and, therefore, expression ofthe ligninase and cellulase fusion proteins are effected by a promoterthat is active in a particular tissue of the plant, e.g., a promoterthat is active primarily in the leaves of a plant. A leaf-specificpromoter that is preferred for transcription (expression at the RNAlevel) is the rice rubisco synthase gene promoter (rbcSP), which has thenucleotide sequence prior to the rbcS gene coding region included in SEQID NO:1. In some embodiments of the present invention, it is desirableto relegate transcription of the heterologous gene expression cassetteto the seeds using a seed-specific promoter. Seed-specific promotersthat are suitable include, but are not limited to, the seed-specificpromoters such as the maize 19 kDa zein (cZ19B1) promoter, the maizecytokinin-induced message (Cim1) promoter, and the maizemyo-inositol-1-phosphate synthase (mi1ps) promoter, which are disclosedin U.S. Pat. No. 6,225,529 to Lappegard et al. Therefore, in theheterologous gene expression cassettes, the nucleotide sequencecomprising rbcS promoters are operably linked to the nucleotidesequences encoding the ligninase and cellulase fusion proteins. Thus, ina transgenic plant of the present invention, transcription of theligninase and cellulase fusion proteins occurs primarily in the leavesof the plant, and because the ligninase and cellulase fusion proteinseach has a signal peptide that directs its transport to plastids, theligninase and cellulase fusion proteins accumulate in the plastids.

In the preferred embodiment of the present invention, the 3′ ends of thenucleotide sequence encoding the above ligninase and cellulase fusionproteins are operably linked to a 3′ noncoding sequence wherein thenoncoding sequence contains a poly(A) cleavage/addition site and otherregulatory sequences which enables the RNA transcribed therefrom to beproperly processed and polyadenylated which in turn affects stability,transport and translation of the RNA transcribed therefrom in the plantcell. Examples of 3′ noncoding sequences include the 3′ noncodingsequence from the potato protease inhibitor II gene, which includesnucleotides 871 to 1241 of SEQ ID NO: 15 (GenBank Accession No. M15186)and the 3′ noncoding sequence from the Agrobacterium nopaline synthasegene, which includes nucleotides 2001 to 2521 of SEQ ID NO:16 (GenBankAccession No. V00087 J01541).

The above heterologous gene expression cassettes can be constructedusing conventional molecular biology cloning methods. In a particularlyconvenient method, PCR is used to produce the nucleotide fragments forconstructing the gene expression cassettes. By using the appropriate PCRprimers, the precise nucleotide regions of the above DNAs can beamplified to produce nucleotide fragments for cloning. By furtherincluding in the PCR primers restriction enzyme cleavage sites which aremost convenient for assembling the heterogenous gene expressioncassettes (e.g., restriction enzyme sites that are not in the nucleotidefragments to be cloned), the amplified nucleotide fragments are flankedwith the convenient restriction enzyme cleavage sites for assembling thenucleotide fragments into heterogenous gene expression cassettes. Theamplified nucleotide fragments are assembled into the heterogeneous geneexpression cassettes using conventional molecular biology methods. Basedupon the nucleotide sequences provided herein, how to construct theheterogenous gene expression cassettes using conventional molecularbiology methods with or without PCR would be readily apparent to oneskilled in the art.

In a further embodiment of the present invention, the transgenic plantcomprises more than one heterogeneous gene expression cassette. Forexample, the transgenic plant comprises a first cassette which containsa DNA encoding a ligninase fusion protein, and one or more cassetteseach containing a DNA encoding a particular cellulase fusion protein.Preferably, both the ligninase and cellulase fusion proteins compriseamino acids of a signal peptide which directs the fusion proteins toplant organelles. In a preferred embodiment, the signal peptide for eachis the rbcS SP or the SKL motif.

In a further still embodiment, the transgenic plant comprises DNAencoding the ligninase fusion protein such as the ckg4 or ckg5 LIP, anendoglucanase fusion protein such as the e1 fusion protein, and acellobiohydrolase fusion protein such as the cbh1 fusion protein. In afurther still embodiment, the transgenic plant comprises DNA encodingthe ligninase fusion protein such as the ckg4 or ckg5 LIP, anendoglucanase fusion protein such as the e1 fusion protein, acellobiohydrolase fusion protein such as the cbh1 fusion protein, abeta-glucosidase, and a glucanhydrolase. Preferably, both the ligninaseand cellulase fusion proteins comprise amino acids of a signal peptidewhich directs the fusion proteins to plant organelles. In a preferredembodiment, the signal peptide for each is the rbcS SP or the SKL motif.

To make the transgenic plants of the present invention, plant materialsuch as meristem primordia tissue (or immature embryo-derived cell andcallus line) is transformed with plasmids, each containing a particularheterogenous gene expression cassette using the Biolistic bombardmentmethod as described in Example 5 and in U.S. Pat. No. 5,767,368 to Zhonget al. Further examples of the Biolistic bombardment method aredisclosed in U.S. application Ser. No. 08/036,056 and U.S. Pat. No.5,736,369 to Bowen at al. Each heterogenous gene expression cassette isseparately introduced into a plant tissue and the transformed tissuepropagated to produce a transgenic plant that contains the particularheterogenous gene expression cassette. Thus, the result is a transgenicplant containing the heterogenous gene expression cassette expressing aligninase such as ckg4 or ckg5, a transgenic plant containing aheterogenous gene expression cassette expressing endoglucanase such ase1, a transgenic plant containing a heterogenous gene expressioncassette expressing a cellobiohydrolase such as cbh1, a transgenic plantcontaining a heterogenous gene expression cassette expressing anexoglucanase such as beta-glucosidase, and a transgenic plant containinga heterogenous gene expression cassette expressing an exoglucanase suchas glucanhydrolase.

Alternatively, transformation of corn plants can be achieved usingelectroporation or bacterial mediated transformation using a bacteriumsuch as Agrobacterium tumefaciens to mediate the transformation of cornroot tissues (see ⁸Valvekens et al. Proc. Nat'l. Acad. Sci. USA. 85:5536-5540 (1988)) or meristem primordia.

In a preferred embodiment of the present invention, the transgenic plantcomprises one or more ligninase fusion proteins and one or morecellulase fusion proteins. Construction of the preferred transgenicplant comprises making first generation transgenic plants as above, eachcomprising a ligninase fusion protein, and transgenic plants as above,each comprising a cellulase fusion protein. After each first generationtransgenic plant has been constructed, progeny from each of the firstgeneration transgenic plants are cross-bred by sexual fertilization toproduce second generation transgenic plants comprising variouscombinations of both the ligninase fusion protein and the cellulasefusion protein.

For example, various combinations of progeny from the first generationtransgenic plants are cross-bred to produce second generation transgenicplants that contain ckg4 and cbh1, e1, beta-glucosidase, or ckg5; secondgeneration transgenic plants that contain ckg5 and cbh1, e1, orbeta-glucosidase; second generation transgenic plants that contain e1 orbeta glucosidase, and a second generation transgenic plant that containse1 and beta-glucosidase.

Progeny of the second generation transgenic plants are cross-bred bysexual fertilization among themselves or with first generationtransgenic plants to produce third generation transgenic plants thatcontain one or more ligninases, one or more cellulases, or combinationsthereof.

For example, cross-breeding a second generation transgenic plantcontaining ckg4 and cbh1 with a second generation transgenic plantcontaining e1 and beta-glucosidase produces a third generationtransgenic plant containing ckg4, cbh1, e1, and beta-glucosidase. Thethird generation transgenic plant can be cross-bred with a firstgeneration transgenic plant containing ckg5 to produce a fourthgeneration transgenic plant containing ckg4, ckg5, cbh1, e1, andbeta-glucosidase.

It will be readily apparent to one skilled in the art that othertransgenic plants with various combinations of ligninases and cellulasescan be made by cross-breeding progeny from particular transgenic plants.⁹Zhang et al, Theor. Appl. Genet. 92: 752-761, (1996), ¹⁰Zhong et al,Plant Physiol. 110: 1097-1107, (1996); and ¹¹Zhong et al, Planta, 187:483-489, (1992) provide methods for making transgenic plants by sexualfertilization.

Alternatively, plant material is transformed as above with a plasmidcontaining a heterologous gene expression cassette encoding theligninase fusion protein. The transgenic plant is recovered from theprogeny of the transformed plant material. Next, plant material from thetransgenic plant is transformed with a second plasmid containing aheterologous gene expression cassette encoding the cellulase fusionprotein and a second selectable marker. The transgenic plant isrecovered from the progeny of the transformed plant material. It will bereadily apparent to one skilled in the art that transgenic plantscontaining any combination of ligninases and cellulases can be made bythe above method.

In a preferred embodiment, the above heterologous gene expressioncassettes further include therein nucleotide sequences that encode oneor more selectable markers which enable selection and identification oftransgenic plants that express the modified cellulase of the presentinvention. Preferably, the selectable markers confers additionalbenefits to the transgenic plant such as herbicide resistance, insectresistance, and/or resistance to environmental stress.

Alternatively, the above transformations are performed byco-transforming the plant material with a first plasmid containing aheterologous gene expression cassette encoding a selectable marker and asecond plasmid containing a heterologous gene expression cassetteencoding a ligninase or cellulase fusion protein. The advantage of usinga separate plasmid is that after transformation, the selectable markercan be removed from the transgenic plant by segregation, which enablesthe selection method for recovering the transgenic plant to be used forrecovering transgenic plants in subsequent transformations with thefirst transgenic plant.

Examples of preferred markers that provide resistance to herbicidesinclude, but are not limited to, the bar gene from Streptomyceshygroscopicus encoding phosphinothricin acetylase (PAT), which confersresistance to the herbicide glufonsinate; mutant genes which encoderesistance to imidazalinone or sulfonylurea such as genes encodingmutant form of the ALS and AHAS enzyme as described by ¹²Lee at al. EMBOJ. 7: 1241 (1988) and ¹³Miki et al., Theor. Appl. Genet. 80: 449 (1990),respectively, and in U.S. Pat. No. 5,773,702 to Penner et al.; geneswhich confer resistance to glycophosphate such as mutant forms of EPSPsynthase and aroA; resistance to L-phosphinothricin such as theglutamine synthetase genes; resistance to glufosinate such as thephosphinothricin acetyl transferase (PAT and bar) gene; and resistanceto phenoxy proprionic acids and cycloshexones such as the ACCAseinhibitor-encoding genes (¹⁴Marshall et al. Theor. Appl. Genet. 83: 435(1992)). The above list of genes which can import resistance to anherbicide is not inclusive and other genes not enumerated herein butwhich have the same effect as those above are within the scope of thepresent invention.

Examples of preferred genes which confer resistance to pests or diseaseinclude, but are not limited to, genes encoding a Bacillus thuringiensisprotein such as the delta-endotoxin, which is disclosed in U.S. Pat. No.6,100,456 to Sticklen et al.; genes encoding lectins, (¹⁵Van Damme etal., Plant Mol. Biol. 24: 825 (1994)); genes encoding vitamin-bindingproteins such as avidin and avidin homologs which can be used aslarvicides against insect pests; genes encoding protease or amylaseinhibitors, such as the rice cysteine proteinase inhibitor (¹⁶Abe etal., J. Biol. Chem. 262: 16793 (1987)) and the tobacco proteinaseinhibitor I (¹⁷Hubb et al., Plant Mol. Biol. 21: 985 (1993)); genesencoding insect-specific hormones or pheromones such as ecdysteroid andjuvenile hormone, and variants thereof, mimetics based thereon, or anantagonists or agonists thereof; genes encoding insect-specific peptidesor neuropeptides which, upon expression, disrupts the physiology of thepest; genes encoding insect-specific venom such as that produced by awasp, snake, etc.; genes encoding enzymes responsible for theaccumulation of monoterpenes, sesquiterpenes, asteroid, hydroxamincacid, phenylpropanoid derivative or other non-protein molecule withinsecticidal activity; genes encoding enzymes involved in themodification of a biologically active molecule (see U.S. Pat. No.5,539,095 to Sticklen et al., which discloses a chitinase that functionsas an anti-fungal); genes encoding peptides which stimulate signaltransduction; genes encoding hydrophobic moment peptides such asderivatives of Tachyplesin which inhibit fungal pathogens; genesencoding a membrane permease, a channel former or channel blocker (forexample cecropin-beta lytic peptide analog renders transgenic tobaccoresistant to Pseudomonas solanacerum) (¹⁸Jaynes et al. Plant Sci. 89: 43(1993)); genes encoding a viral invasive protein or complex toxinderived therefrom (viral accumulation of viral coat proteins intransformed cells of some transgenic plants impart resistance toinfection by the virus the coat protein was derived as shown by ¹⁹Beachyat al. Ann. Rev. Phytopathol. 28: 451 (1990); genes encoding aninsect-specific antibody or antitoxin or a virus-specific antibody(²⁰Tavladoraki et al. Nature 366: 469 (1993)); and genes encoding adevelopmental-arrestive protein produced by a plant, pathogen orparasite which prevents disease. The above list of genes which canimport resistance to disease or pests is not inclusive and other genesnot enumerated herein but which have the same effect as those above arewithin the scope of the present invention.

Examples of genes which confer resistance to environmental stressinclude, but are not limited to, mt1d and HVA1, which are genes thatconfer resistance to environmental stress factors; rd29A and rd19B,which are genes of Arabidopsis thaliana that encode hydrophilic proteinswhich are induced in response to dehydration, low temperature, saltstress, or exposure to abscisic acid and enable the plant to toleratethe stress (²¹Yamaguchi-Shinozaki et al., Plant Cell 6: 251-264 (1994)).Other genes contemplated can be found in U.S. Pat. Nos. 5,296,462 and5,356,816 to Thomashow. The above list of genes, which can importresistance to environmental stress, is not inclusive and other genes notenumerated herein but which have the same effect as those above arewithin the scope of the present invention.

Thus, it is within the scope of the present invention to providetransgenic plants which express one or more ligninase fusion proteins,one or more cellulase fusion proteins, and one or more of anycombination of genes which confer resistance to an herbicide, pest, orenvironmental stress.

In particular embodiments of the present invention, the heterologousgene expression cassettes can further be flanked with DNA containing thematrix attachment region (MAR) sequence. While use of MAR in the presentinvention is optional, it can used to increase the expression level oftransgenes, to get more reproducible results, and to lower the averagecopy number of the transgene (²²Allen et al., The Plant Cell 5: 603-613(1993); ²³Allen et al., The Plant Cell 8: 899-913 (1996); ²⁴Mlynarova etal., The Plant Cell 8: 1589-1599 (1996)).

To degrade the lignocellulose in the leaves and stalks of the transgenicplants of the present invention, the transgenic plant is ground up toproduce a plant material using methods currently available in the art todisrupt a sufficient number of the plant organelles containing theligninase and cellulase therein. The ligninase and cellulase degrade thelignocellulose of the transgenic plant into fermentable sugars,primarily glucose, and residual solids. The fermentable sugars are usedto produce ethanol or other products.

The transgenic plants can be processed to ethanol in an improvement onthe separate saccharification and fermentation (SHF) method (¹Wilke etal., Biotechnol. Bioengin. 6: 155-175 (1976)) or the simultaneoussaccharification and fermentation (SSF) method disclosed in U.S. Pat.No. 3,990,944 to Gauss et al. and U.S. Pat. No. 3,990,945 to Huff et al.The SHF and SSF methods require pre-treatment of the plant materialfeedstock with dilute acid to make the cellulose more accessiblefollowed by enzymatic hydrolysis using exogenous cellulases to produceglucose from the cellulose, which is then fermented by yeast to ethanol.In some variations of the SHF or SSF methods, the plant material ispre-treated with heat or with both heat and dilute acid to make thecellulose more accessible.

An SHF or SSF method that uses the transgenic plant material of thepresent invention as the feedstock is an improvement over the SHF or SSFmethod because the transgenic plant material contains its own cellulasesand ligninases or cellulases. Therefore, exogenous ligninases and/orcellulases do not need to be added to the feedstock. Furthermore,because particular embodiments of the transgenic plant material produceligninase, the need for pre-treatment of the plant material in thoseembodiments before enzymatic degradation is not necessary. In a furtherimprovement over the SHF method, the transgenic plant material is mixedwith non-transgenic plant material and the mixture processed to ethanol.

The following examples are intended to promote a further understandingof the present invention.

Example 1

This example shows the construction of plasmids comprising aheterologous gene expression cassette comprising a DNA encoding acellulase fusion protein and a heterologous gene expression cassettecomprising a DNA encoding the bar gene (Table 1).

TABLE 1 Construct Plasmid features 1 rbcSP/e1/pin 3′//Act1 P/bar/nosrbcSP leaf-specific promoter 3′ driving cellulase cDNA of A.cellulolyticus 2 rbcSP/cbh1/pin 3′//Act1 rbcSP leaf-specific promoterP/bar/nos 3′ driving cellulase cDNA of T. reesi 3 rbcSP/rbcS SP/e1/pin3′//Act1 The rbcS SP targets cellulase P/bar/nos 3′ of A. cellulolyticusinto maize chloroplasts 4 rbcSP/rbcS SP/cbh1/pin 3′// Act1 The rbcS SPtargets cellulase P/bar/nos 3′ of T. reesi into maize chloroplasts

Abbreviations:

The term “rbcSP” means the rice rubisco rbcS promoter region. The rbcSPis a leaf-specific promoter that limits transcription of rbcS to theleaves (²⁵Schaeffer and Sheen, Plant Cell 3: 997-1012 (1991)). Thenucleotide sequence for the rbcS promoter region is set forth in SEG IDNO:1.

The term “e1” means the cDNA isolated from Acidothermus cellulolyticuswhich encodes the cellulase E1 beta-1,4-endoglucanase precursor. Thenucleotide sequence for the gene encoding e1 is set forth in SEQ IDNO:4. In this example, the codons for the 41 amino acid leader sequence(nucleotides 824 to 946 of SEQ ID NO:4) are removed.

The term “cbh1” means the cDNA isolated from Trichoderma reesi thatencodes the cellulase cellobiohydrolase. The nucleotide sequence for thegene encoding cbh1 is set forth in SEQ ID NO:10. In this example, thecodons for the 54 amino acid leader sequence (nucleotides 210 to 671 ofSEQ ID NO:10) are removed.

The term “pin3′” means the potato protease inhibitor II-chloramphenicolacetyltransferase gene's 3′ untranslated sequence which containstranscription termination signals (²⁶Thornburg et al., Proc. Natl. Acad.Sci. USA 84: 744-748 (1987)). The pin3′ untranslated sequence includesnucleotides 882 to 1241 of the nucleotide sequence set forth in SEQ IDNO: 15.

The term “bar” means the phosphinothricin acetyl transferase gene(²⁷Thompson et al., EMBO J. 6: 2519-2523 (1987)). The bar gene is aselectable marker for herbicide resistance. The 5′ end of bar isoperably linked to the rice actin 1 gene promoter which has been shownto operable in maize (¹⁰Zhong et al., Plant Physiology 110: 1097-1107(1996); ⁹Zhang et al., Theor. Appl. Genet. 92: 752-761 (1996); ²⁸Zhanget al., Plant Science 116: 73-84 (1996)). The 3′ end of bar is operablylinked to the nos 3′ untranslated sequences. The nucleotide sequence ofthe bar gene is set forth in SEQ ID NO:18 (GenBank Accession No.X05822), which encodes the bar having the amino acid sequence fromnucleotides 160 to 711.

The term “Act1 P” means the rice Act1 gene promoter which furtherincludes the 5′ intron region (²⁹McElroy et al., Mol. Gen. Genet. 231:150-160 (1991)). The sequence of the Act1 gene and its promoter is setforth in SEQ ID NO:19 (GenBank Accession No. X63830).

The term “nos3′” means the 3′ untranslated sequence from theAgrobacterium nopaline synthase gene encoding nopaline synthase of theamino acid sequence as set forth in SEQ ID NO:17 which includesnucleotides 2002 to 2521 of SEQ ID NO:16 (GenBank Accession No. V00087J01541). The Nos3′ sequence contains transcription termination signals.

The term “rbcS SP” means the rice rubisco small subunit signal peptidewhich consists of 47 codons encoding the peptide with the amino acidsequence set forth in SEQ ID NO:2. The rbcS SP directs the translocationof the rbcS small subunit or any polypeptide to which it is attached tothe chloroplasts (³⁰Loza-Tavera et al., Plant Physiol. 93: 541-548(1990)).

Construct 1 contains the rice rubisco rbcS leaf-specific promoter whichlimits expression of the cellulase encoded by e1 to the cells of theleaves of the maize plant.

Construct 2 contains the rice rubisco rbcS leaf-specific promoter whichlimits expression of the cellulase encoded by cbh1 to the cells of theleaves of the maize plant.

Construct 3, which is shown in FIG. 1, is like construct 1 except thatDNA encoding the rbcS SP signal peptide is operably linked to the 5′ endof the e1, and construct 4, which is shown in FIG. 2, is like construct2 except that DNA encoding the rbcS SP signal peptide is operably linkedto the 5′ end of cbh1. Therefore, expression of cellulase from construct3 or 4, which is limited to the cells of the leaves, directed to thechloroplasts in the cells. All of the above constructs are adjacent to aheterologous gene expression cassette containing the bar gene operablylinked to the Act1 promoter.

Construction of plasmid rbcSP/rbcS SP/cbh1//pin3′//Act1 P/bar/nos3′. Thestarting plasmid was pBR10-11 which contained the cry1A(b) gene upstreamof the pin3′. Between the cry1A(b) and the pin3′ is a DNA polylinkercontaining in the following order a SmaI, BamHI, SpeI, XbaI, NotI, andEagI restriction enzyme recognition site. The plasmid pBR10-11(available from Silan Dai and Ray Wu, Department of Molecular Biologyand Genetics, Biotechnology Building, Cornell University, Ithaca, N.Y.14853-2703) was digested with restriction enzymes SpeI and XbaI toproduce a 9.2 kb DNA fragment. The 9.2 kb DNA fragment(pBR10-11/SpeI/XbaI/9.2 kb fragment) was purified by agarose gelelectrophoresis.

The plasmid pB210-5a (available from William S. Adney, Mike Himmel, andSteve Thomas, National Renewable Energy Laboratory, 1670 Cole Boulevard,Golden Colo. 80401) containing the cbh1 gene from Trichoderma reesei(Trichoderma longibrachiatum) was digested with SpeI and XbaI. Thedigested plasmid was electrophoresed on an agarose gel and a 1.8 kbfragment (pB210-5a/SpeI/XbaI/1.8 kb fragment containing cbh1) waspurified from the gel.

The above 9.2 kb and the 1.8 kb DNA fragments were ligated togetherusing T4 DNA ligase to make plasmid “pBR10-11-cbh1” which was used totransform E. coli XL1 Blue. Transformed bacteria containing plasmidpBR10-11-cbh1 were identified by plating on LB agar gels containingampicillin.

The plasmid pBR10-11-cbh1 was digested with SmaI and PstI. The PstI endwas made blunt with mung bean exonuclease. The digested plasmid waselectrophoresed on an agarose gel and the 2.8 kb DNA fragment containingcbh1 and pin3′ was purified from the gel. The purified DNA fragment wasdesignated “cbh1-pin3′/blunt-ended.”

The plasmid pDM302 (³¹Cao at al., Plant Cell Reports 11: 586-591(1992)), shown in FIG. 3, containing upstream of a ClaI site, a genecassette consisting of the bar gene flanked by an upstream Act1 promoterand a downstream nos3′, was digested with ClaI. The ClaI ends of thedigested plasmid were made blunt with Taq DNA polymerase and thedigested plasmid electrophoresed on an agarose gel. The digested plasmidwas designated “pDM302/ClaI/blunt-ended.”

The pDM302/ClaI/blunt-ended plasmid and the cbh1-pin3′/blunt-ended DNAfragment were ligated together using T4 DNA ligase to make plasmid“pDM302-cbh1-pin3′” which was used to transform E. coli XL1Blue.Transformed bacteria containing plasmid pDM302-cbh1-pin3′ wereidentified by plating on LB agar gels containing ampicillin.

Plasmid pDM302-cbh1-pin3′ was digested with SpeI, the ends made bluntwith Taq DNA polymerase, and purified by agarose gel electrophoresis.The purified DNA fragment was designated“pDM302-cbh1-pin3′/SpeI/blunt-ended.”

Plasmid pRR1 (available from Silan Dai and Ray Wu, Department ofMolecular Biology and Genetics, Biotechnology Building, CornellUniversity, Ithaca, N.Y. 14853-2703), which contains the rice rbcS smallsubunit gene, was digested with PstI. The rbcS promoter is flanked byPstI sites. The PstI ends were made blunt with mung bean nuclease andthe 2 kb DNA fragment (rice rbcS/PstI/blunt-ended) containing thepromoter was purified by agarose gel electrophoresis.

Rice rbcSP/PstI/blunt-ended and plasmid pDM-cbh1-pin3′/SpeI/blunt-endedwere ligated using T4 DNA ligase to makerbcSP/cbh1/pin3′//Act1P/bar/nos3′ which was then used to transform E.coli XL Blue. Transformed bacteria containing plasmidrbcSP/cbh1/pin3′//Act1P/bar/nos3′ were identified by plating on LB agargels containing ampicillin.

PCR was used to insert NotI sites intorbcSP/cbh1/pin3′//Act1P/bar/nos3′. These sites were used to insert therice rubisco signal peptide in place of the cbh1 signal peptide. ThepRRI plasmid was the source of the rice rubisco signal peptide. It wasalso the used as a PCR template to produce the PCR product containingthe rice rubisco signal peptide flanked by NotI cohesive termini. Therice rubisco signal peptide and the rbcSP/cbh1/pin3′//Act1P/bar/nos3′plasmid were ligated together using T4 DNA ligase to make rbcSP/rbcSSP/cbh1/pin3′//Act1P/bar/nos3′ which was then used to transform E. coliXL Blue. Transformed bacteria containing plasmid rbcSP/rbcS

SP/cbh1/pin3′//Act1P/bar/nos3′ were identified by plating on LB agargels containing ampicillin.

Construction of plasmid rbcSP/rbcS SP/e1/pin3′//Act1P/bar/nos3′. PlasmidpMPT4-5 (available from William S. Adney, Mike Himmel, and Steve Thomas,national Renewable Energy laboratory, 1670 Colorado Boulevard, Golden,Colo. 80401) contains the e1 gene encoding endoglucanase I fromAcidothermus cellulolyticus as a 3.7 kb PvuI DNA fragment cloned intopGEM7 (Promega Corporation, Madison, Wis.). PCR was used to produce aDNA fragment containing the e1 gene flanked by AscI recognition sites.Plasmid rbcSP/cbh1/pin3Y/Act1P/bar/nos3′ was also mutagenized by PCR tointroduce AscI sites flanking the cbh1 gene. Next, the plasmidrbcSP/cbh1/pin3′//Act1P/bar/nos3′ was digested with AscI and the plasmidfree of the cbh1 gene was purified by agarose gel electrophoresis. TheAscI flanked e1 gene was ligated using T4 DNA ligase into therbcSP/cbh1/pin3′//Act1P/bar/nos3′ free of the cbh1 gene to produceplasmid rbcSP/e1/pin37/Act1P/bar/nos3′, which then used to transform E.coli. XL Blue. Transformed bacteria containing plasmidrbcSP/e1/pin3′//Act1P/bar/nos3′ were identified by plating on LB agargels containing ampicillin.

PCR was used to insert NotI sites into rbcSP/e1/pin3′//Act1P/bar/nos3′.These sites were used to insert the rice rubisco signal peptide in placeof the cbh1 signal peptide. The pRRi plasmid was the source of the ricerubisco signal peptide. It was also the used as a PCR template toproduce the PCR product containing the rice rubisco signal peptideflanked by NotI cohesive termini. The rice rubisco signal peptide andthe rbcSP/e1/pin3′//Act1P/bar/nos3′ plasmid were ligated together usingT4 DNA ligase to make rbcSP/rbcS SP/e1/pin3′//Act1P/bar/nos3′ which wasthen used to transform E. coli XL Blue. Transformed bacteria containingplasmid rbcSP/rbcS SP/e1/pin3′//Act1P/bar/nos3′ were identified byplating on LB agar gels containing ampicillin.

Both heterologous gene expression cassettes are contiguous and thecontiguous cassettes can be flanked by MAR sequences.

Example 2

This example shows the construction of plasmids comprising aheterologous gene expression cassette comprising a DNA encoding acellulase fusion protein. The plasmid constructs are shown in Table 2.

TABLE 2 Construct Plasmid features 1 rbcSP/cbh1/pin 3′ rbcSPleaf-specific promoter driving cellulase cDNA of T. reesei 2 rbcSP/rbcSSP/cbh1/pin 3′ The rbcS SP targets cellulase of T. reesi into maizechloroplasts 3 rbcSP/rbcS SP/syn-cbh1/pin 3′ The rbcS SP targetsmodified cellulase of T, reesei into maize chloroplasts 4CaMv35s/SSU/e1/nos3′ The SSU targets the cellulase of A. cellulolyticusinto maize chloroplasts 5 CaMv35s/VSP/e1/nos3′ The VSP targets thecellulase of A. cellulolyticus into maize apoplasts 6 CaMv35s/e1/nos3′No signal peptide

Abbreviations:

The term “syn-cbh1” refers to a cbh1 gene that has been codon-modifiedfor use in transformation of tobacco plants.

The term “CaMV3Ss” refers to the cauliflower mosaic virus promoter.

The term “SSU” refers to the glycine max rbcS signal peptide. Glycinemax is a soybean and not a rice variety.

The term “VSP” refers to the soybean vegetative storage protein betasignal peptide.

The remainder of the terms in Table 2 are the same as those for table 1.

Construct 1, which is shown in FIG. 4, is plasmid pSMF13 which isplasmid pSK (Stratagene, La Jolla, Calif.) which contains cbh1 operablylinked to the rice rubisco rbcS leaf-specific promoter which limitsexpression of the cellulase encoded by cbh1 to the cells of the leavesof the maize plant.

Construct 2, which is shown in FIG. 5, is plasmid pSF15 which is plasmidpSK which contains cbh1 operably linked to the rice rubisco rbcSleaf-specific promoter which limits expression of the cellulase encodedby cbh1 to the cells of the leaves of the maize plant and a DNA encodingthe rbcS SP which targets the cellulase to the chloroplasts.

Construct 3, which is shown in FIG. 6, is like construct 2 except thatthe cbh1 has been modified to decrease the GC content of the cbh1 to anamount similar to the GC content of the tobacco plant genome. Thenucleotide sequence of the modified cbh1 (syn-cbh1) in plasmid pBI221 isset forth in SEQ ID NO:20.

Construct 4, which is shown in FIG. 7, is plasmid pTZA8 which is plasmidpBI121 which contains the caMV35s promoter, which is a constitutivepromoter that is active in most plant tissues, to drive expression of e1which is operably linked to a DNA encoding the SSU signal peptide whichtargets the cellulase to the chloroplasts.

Construct 5, which is shown in FIG. 8, is plasmid pZA9 which is similarto construct 4 except the signal peptide is encoded by DNA encoding theVSP signal peptide which targets the cellulase to the apoplasts.Construct 6, which is shown in FIG. 9, is plasmid pZA10 which is similarto construct 4 or 5 except that e1 is not operably linked to a DNAencoding a signal peptide that targets the cellulase to a plantorganelle.

The constructs were prepared as follows.

First, the plasmid pRR1, which contains the rice rbcS gene was obtainedfrom Ray Wu and Silan Dai, Cornell University. The rice rubisco (rbcS)small subunit was cleaved from pRR1 using EcoRI and EcoRV restrictionsites to release a 2.1 kb DNA fragment containing the rbcS. The 2.1 kbDNA fragment was ligated into the plasmid pSK between the EcoR1 andEcoRV sites to produce plasmid pSMF8. The 2.1 kb DNA fragment providedthe promoter for the cbh1 constructs below.

Next, the cbh1 gene was cloned downstream of rbcS promoter in plasmidpSMF8. First, a 1.7 kb DNA fragment containing the cbh1 gene fromTrichoderma reesei was isolated from plasmid pB210-5A (available fromWilliam S. Adney, Mike Himmel, and Steve Thomas, National RenewableEnergy Laboratory; described in ³²Shoemaker et al., Bio/Technology 1:691-696 (1983)) by digesting with SalI and XhoI. The ends of the 1.7 kbDNA fragment were made blunt end using DNA polymerase I (largefragment). The blunt-ended DNA fragment was cloned into plasmid pSMF8,which had been digested with BamHI and the ends made blunt with DNApolymerase I, to make plasmid pSMF9.

Next, to complete the heterologous gene expression cassette, the pin3′transcription termination nucleotide sequence was inserted at the 3′ endof the cbh1 gene in plasmid pSMF9. The pin3′ transcription terminationnucleotide sequence was cleaved from pBR10-11 with PstI. However, toremove the pin3′ transcription termination nucleotide sequence frompBR10-11, a PstI site had to be introduced upstream of the pin3′transcription termination nucleotide sequence.

To generate the PstI site upstream of the pin3′ transcriptiontermination nucleotide sequence in pBR10-11, the pBR10-11 was digestedwith NotI and XhoI and a 70 bp multi-cloning site nucleotide sequence,which had been isolated from the pSK vector by digesting with NotI andXhoI, was cloned between the NotI and XhoI sites of the pBR10-11 toproduce plasmid pSMF11. The pin3′ transcription termination nucleotidesequence was then removed from pSMF11 by digesting with PstI to producea 1 kb DNA fragment which was then cloned into the PstI site of pSK,which had been digested with PstI, to produce plasmid pSMF12. PSMF12 wasthen digested with NotI to produce a 1 kb DNA fragment containing thepin3′ transcription termination nucleotide sequence. The 1 kb DNAfragment cloned into the NotI site downstream of the cbh1 gene in pSMF9,which had been digested with NotI, to produce plasmid pSMF13 (construct1 in Table 2).

Next, a DNA encoding a signal peptide which targets proteins to which itis attached to the chloroplasts was inserted upstream of the cbh1 and inthe same reading frame as the cbh1. Thus, a fusion protein is producedfrom translation of RNA transcribed from the cbh1 DNA linked to the DNAencoding the signal peptide. DNA encoding the signal peptide (SP) wasisolated from the rbcS in the pRR1 plasmid. Because there were noconvenient restriction enzyme sites available which flanked the DNAencoding the SP for cloning, it was planned to PCR amplify that regioncontaining the DNA encoding the SP using PCR primers with PCR primersthat contained convenient restriction enzyme sites for cloning. At theend of the rbcS promoter pSMF13 is a unique AvrII site and upstream ofthe first ATG of the cbh1 gene is a unique BsrGI. A DNA encoding the SPthat was flanked with an AvrII site on one end and a BsrGI site on theopposite end would be able to be cloned between the AvrII and BsrGIsites in PSMF13. That would place the DNA encoding the SP between therbcS promoter and the cbh1 gene and would enable a fusion proteincontaining the SP fused to the cellulase.

Therefore, PCR primers were synthesized using DNA sequences for theAvrII and BsrGI sites and the SP DNA sequences. The upstream PCR primer(SP1F) had the nucleotide sequence 5′-CCGCCIAGGCGCATGGCCCCCTCCGT-3′ (SEQID NO:21) and the downstream PCR primer (SP3R) had the nucleotidesequence 5′-CGCTGTACACGCACCTGATCCTGCC-3′ (SEQ ID NO:22). Plasmid pRR1encoding the SP was PCR amplified with the PCR primers and the 145 bpamplified product was purified using 2% agarose gel. The purified 145 bpproduct was sequenced to confirm that the 145 bp amplified productcontained the SP nucleotide sequences. The amplified product wasdigested with AvrII and BsrGI and cloned between the AvrII and BsrGIsites of pSMF13 digested with AvrII and BsrGI to produce plasmid pSMF14.

To produce pSMF15 which contains a cbh1 gene codon-modified to decreasethe GC content of the cbh1 gene to an amount similar to the GC contentof the tobacco genome, a synthetic cbh1 (syn-cbh1) gene was obtainedfrom plasmid pZD408 (available from Ziyu Dai, Pacific Northwest nationalLaboratory, 902 Battelle Boulvard, Richland, Wash. 99352). The syn-cbh1is a cbh1 which had been codon-modified for use in tobacco planttransformations. The nucleotide sequence of syn-cbh1 is set forth in SEQID NO:20. Plasmid pZD408 was linearized with NcoI and the ends madeblunt. Then, the blunt-ended pZD408 was digested with HindIII to removethe CaMV35S promoter. A 4.5 kb DNA fragment containing the syn-cbh1 wasisolated from the CaMV35S promoter by agarose gel electrophoresis. The4.5 kb DNA fragment was dephosphorylated and the DNA fragment containinga blunt end and a HindIII end was named pZD408B.

Plasmid pSMF14 was digested with BsrGI, the BsrGI ends made blunt, andthen pSMF14 was digested with HindIII to produce a DNA fragmentcontaining the rbcS promoter with the DNA encoding the SP flanked by ablunt end and a HindIII end. The DNA fragment was purified by agarosegel electrophoresis and ligated to the pZD408B DNA fragment to produceplasmid pSMF15 (construct 3 of Table 2).

The heterologous gene expression cassettes are contiguous can be flankedby MAR sequences.

Example 3

This example shows the construction of plasmids comprising aheterologous gene expression cassette comprising a DNA encoding aligninase fusion protein and a heterologous gene expression cassettecomprising a DNA encoding the bar gene. The constructs are shown inTable 3.

TABLE 3 Construct Plasmid features 1 rbcSP/ckg4/pin 3′//Act1 rbcSPleaf-specific P/bar/nos 3′ promoter driving ckg4 cDNA of P.chrysosporium 2 rbcSP/ckg5/pin 3′//Act1 rbcSP leaf-specific P/bar/nos 3′promoter driving ckg5 cDNA of P. chrysosporium 3 rbcSP/rbcS SP/ckg4/pin3′//Act1 The rbcS SP targets ckg4 P/bar/nos 3′ into maize chloroplasts 4rbcSP/rbcS SP/ckg5/pin 3′// The rbcS SP targets ckg5 Act1 P/bar/nos 3′into maize chloroplasts

Abbreviations:

The terms “ckg4” and “ckg5” mean the ligninase cDNAs isolated from thebasidiomycete Phanerochaete. chrysosporium, SEQ ID NO:11 and SEQ IDNO:13, respectively. The codons for the 28 amino acid leader are deletedso that the expressed gene product remains inside the cells.

The remainder of the terms in Table 3 are the same as those for Table 1.All plasmid constructs contain the selectable marker gene (bar) drivenby the rice actin 1 gene promoter. The rice actin gene and its promoterare disclosed in U.S. Pat. No. 5,641,876 to McElroy et al.

Construct 1 contains the rice rubisco rbcS leaf-specific promoter whichlimits expression of the ligninase encoded by ckg4 to the cells of theleaves of the maize plant.

Construct 2 contains the rice rubisco rbcS leaf-specific promoter whichlimits expression of the ligninase encoded by ckg5 to the cells of theleaves of the maize plant.

Construct 3, which is shown in FIG. 10, contains the rice rubisco rbcSleaf-specific promoter which limits expression of the ligninase encodedby ckg4 to the cells of the leaves of the maize plant and furthercontains DNA encoding the rbcS SP which targets the ligninase to thechloroplasts.

Construct 4, which is shown in FIG. 11, contains the rice rubisco rbcSleaf-specific promoter which limits expression of the ligninase encodedby ckg5 to the cells of the leaves of the maize plant and furthercontains DNA encoding the rbcS SP which targets the ligninase to thechloroplasts. All of the above constructs are adjacent to a heterologousgene expression cassette containing the bar gene operably linked to theAct1 promoter. Both heterologous gene expression cassettes arecontiguous and the contiguous cassettes can be flanked by MAR sequences.

Example 4

This example shows the construction of plasmids comprising aheterologous gene expression cassette comprising a DNA encoding aligninase fusion protein. The constructs are shown in Table 4.

TABLE 4 Construct Plasmid features 1 rbcSP/ckg4/pin 3′ rbcSPleaf-specific promoter driving ckg4 cDNA of P. chrysosporium 2rbcSP/ckg5/pin 3′ rbcSP leaf-specific promoter driving ckg5 cDNA of P.chrysosporium 3 rbcSP/rbcS SP/ckg4/pin 3′ The rbcS SP targets ckg4 intomaize chloroplasts 4 rbcSP/rbcS SP/ckg5/pin 3′ The rbcS SP targets ckg5into maize chloroplasts

The terms in table 4 are the same as those for Tables 1 and 3.

Construct 1, which is shown in FIG. 12, is plasmid pSMF18 which isplasmid pSK which contains the rice rubisco rbcS leaf-specific promoterwhich limits expression of the ligninase encoded by ckg4 to the cells ofthe leaves of the maize plant.

Construct 2, which is shown in FIG. 13, is plasmid pSMF19 which isplasmid pSK which contains the rice rubisco rbcS leaf-specific promoterwhich limits expression of the ligninase encoded by ckg5 to the cells ofthe leaves of the maize plant.

Construct 3, which is shown in FIG. 14, is plasmid pMSF16 which isplasmid pSK which contains the rice rubisco rbcS leaf-specific promoterwhich limits expression of the ligninase encoded by ckg4 to the cells ofthe leaves of the maize plant and further contains DNA encoding the rbcSSP which targets the ligninase to the chloroplasts.

Construct 4, which is shown in FIG. 15, is plasmid pSMF17 which isplasmid pSK which contains the rice rubisco rbcS leaf-specific promoterwhich limits expression of the ligninase encoded by ckg5 to the cells ofthe leaves of the maize plant and further contains DNA encoding the rbcSSP which targets the ligninase to the chloroplasts. The aboveheterologous gene expression cassettes can be flanked by MAR sequences.

The ligninase constructs shown in Table 4 are prepared as describedbelow.

Two plasmids, pCLG4 and pCLG5, the former containing a cDNA cloneencoding the ligninase gene ckg4 and the latter containing a cDNA cloneencoding the ckg5 were obtained from Dr. C. Adinarayana Reddy,Department of Microbiology and Public Health, Michigan State Universityand described in ⁶de Boer et al., Gene 6: 93-102 (1987), ⁷Corrigendum inGene 69: 369 (1988). These ligninase cDNA clones were prepared from awhite-rot filamentous fungus (Phanerochaete chrysosporium). The cDNAsfor ckg4 and ckg5 had each been cloned into the PstI site of the pUC9plasmid to make pCLG4 and pCLG5, respectively. The codons for the28-amino acid leader sequence is deleted from both cDNAs before cloningso that expressed gene product remains inside the cell.

Plasmid pSMF16 is made as follows. The ckg4 gene is removed from pCLG4by digesting the plasmid with the restriction enzymes XbaI and BstEII toproduce a 1.2 kb DNA fragment containing the ckg4 without the nucleotidesequence encoding the transit peptide. The BstEII removes the nucleotidesequences encoding the transit peptide of the ligninase.

The ends of the DNA fragment containing the ckg4 gene are made blunt andthe blunt-ended DNA fragment is ligated into pSMF14 in which the cbh1has been removed by digesting with BsrGI and XhoI and the ends madeblunt to produce pSMF16.

Plasmid pSMF18 is made as follows. The nucleotide sequence encoding therbcS signal peptide and cbh1 are removed from pSMF14 by digesting pSMF14with AvrII and XhoI instead of BsrGI and XhoI. The ends of the digestedpSMF14 are made blunt and the blunt-ended DNA fragment containing theckg4 gene, prepared as above, is ligated into the digested pSMF14 tomake plasmid pSMF18.

Plasmid pSMF17 is made as follows. The ckg5 gene is removed from pCLG5by digesting the plasmid with the restriction enzymes XbaI and EagI toproduce a 1.2 kb DNA fragment containing the ckg5 without the nucleotidesequence encoding the transit peptide. The EagI removes the nucleotidesequences encoding the transit peptide of the ligninase.

The ends of the DNA fragment containing the ckg5 are made blunt and theblunt-ended DNA fragment is ligated into pSMF14 in which the cbh1 hasbeen removed by digesting with BsrGI and XhoI and the ends made blunt toproduce pSMF17.

Plasmid pSMF19 is made as follows. The nucleotide sequence encoding therbcS signal peptide and cbh1 are removed from pSMF14 by digesting pSMF14with AvrII and XhoI instead of BsrGI and XhoI. The ends of the digestedpSMF14 are made blunt and the blunt-ended DNA fragment containing theckg5 gene, prepared as above, is ligated into the digested pSMF14 tomake plasmid pSMF19.

Example 5

This example shows the transformation of maize multi-meristem primordiavia Biolistic bombardment with the plasmid constructs of Examples 1-4,regeneration of the transgenic plants, confirmation of the integrationof the plasmid constructs into the plant genome, and confirmation of theexpression of the cellulase or ligninase fusion proteins in thetransgenic plant. For transformations with the constructs of Examples 2and 4, which do not contain a selectable marker, a selectable markercomprising the bar gene in the plasmid pDM302 (³¹Cao et al., Plant CellReports 11: 586-591 (1992)) is cotransfected into the cells with theplasmid containing the ligninase or cellulase heterologous geneexpression cassette.

Maize seeds have been germinated in Murashige and Skoog (MS) medium(³³Murashige and Skoog, Physiol. Plant 15: 473-497 (1962)) supplementedwith the appropriate growth regulators (³⁴Zhong et al., Planta 187:490-497 (1992)). Shoot meristems have been dissected and cultured for2-3 weeks until an initial multiplication of meristem have been producedfor bombardment.

The multi-meristem primordia explants (or immature embryo-derived celland callus lines) are bombarded with tungsten particles coated withparticular plasmids of Example 1 or 3 or with particular plasmids ofExample 2 or 4 along with the plasmid containing the heterogenous geneexpression cassette containing the bar gene. The bombarded explants aregently transferred onto meristem multiplication medium for furthermultiplication, about 6 to 8 more weeks. This step is required to reducethe degree of chimerism in transformed shoots prior to their chemicalselection. Shoots are transferred to the above medium containing 5 to 10mg per liter glufosinate ammonium (PPT) selectable chemical for another6 to 8 weeks. Chemically selected shoots are rooted in rooting mediumcontaining the same concentration of PPT. Plantlets are transferred topots, acclimated, and then transferred to a greenhouse.

When the plantlets or shoots are small, the quantity of transgenic plantmaterial is insufficient for providing enough DNA for Southern blothybridization; therefore, polymerase chain reaction (PCR) is used toconfirm the presence of the plasmid constructs the plantlets. Theamplified DNA produced by PCR is resolved by agarose or acrylamide gelelectrophoresis, transferred to membranes according standard Southerntransfer methods, and probed with the appropriate DNA construct orportion thereof according to standard Southern hybridization methods.Those shoots or plantlets which show they contain the construct in itsproper form are considered to have been transformed. The transformedshoots or plantlets are grown in the greenhouse to produce sufficientplant material to confirm that the plasmid constructs has been properlyintegrated into the plant genome. To confirm proper integration of theplasmid constructs into the plant genome, genomic DNA is isolated fromthe greenhouse grown transgenic plants and untransformed controls andanalyzed by standard Southern blotting methods as in ¹⁰Zhong et al.,Plant Physiology 110: 1097-1107 (1996); ⁹Zhang et al., Theor. Appl.Genet. 92: 752-761 (1996); ²⁸Zhang et al., Plant Science 116: 73-84(1996); and, ³⁵Jenes et al., In Transgenic Plants. Vol. 1. Kung, S-D andWu, R (eds.). Academic Press, San Diego, Calif. pp. 125-146 (1992).

To confirm expression of the ligninase or cellulase fusion protein,total cellular RNA is isolated from the greenhouse grown plant tissuesas described in ¹⁰Zhong et al., Plant Physiology 110: 1097-1107 (1996).The mRNA encoding the cellulase or ligninase fusion protein is detectedby RNA Northern blot analysis using the same probes used for theSouthern blot analyses. Briefly, the RNA is electrophoresed on adenaturing formaldehyde agarose gel, transferred to nitrocellulose ornylon membranes, hybridized to the appropriate ligninase or cellulaseprobe, and then exposed to X-ray autoradiology film. The hybridizationbands are scanned using a densitometer which enables determination ofthe expression level of the specific mRNA.

Translation of the mRNA is confirmed by Western blot analysis accordingto the standard methods of ³⁶Towbin et al., Proc. Natl. Acad. Sci. USA76: 4350 (1979) and ³⁷Burnette, Anal. Biochem. 112: 195 (1981) usingantibodies specific for ligninase or cellulase.

Example 6

Transgenic maize containing both a ligninase and a cellulase fusionprotein is made by crossing-breeding the abovementioned transgenicplants one of which contains cbh1 or e1 stably integrated into thegenome and the other of which contains ckg4 or ckg5 stably integratedinto the genome using the method provided in (⁹Zhang et al, Theor. Appl.Genet. 92: 752-761, (1996); ¹⁰Zhong et al, Plant Physiol. 110:1097-1107, (1996); ¹¹Zhong et al, Planta, 187: 483-489, (1992)).Transgenic plants that carry a low copy number of the DNA encoding theligninase or cellulase fusion proteins are used for cross-breeding.

Briefly, transgenic maize plants that produce the ligninase fusionprotein are made as disclosed in Example 5 to make a first transgenicplant and transgenic maize plants that produce the cellulase fusionprotein are made as disclosed in Example 5 to make a second transgenicplant. The first and second transgenic plants are cross-pollinated tocreate a transgenic plant which produces both a ligninase and acellulase fusion protein. The progeny are analyzed for homozygosity andtransgenic plants that are homozygous for both the ligninase genecassette and the cellulase gene cassette are selected for furtherpropagation for seeds.

The progeny in the above crosses are used in subsequent crosses toproduce transgenic maize with both ligninase gene cassettes and one,two, or three cellulase gene cassettes or transgenic maize with two orthree cellulase gene cassettes and one ligninase gene cassette.

Example 7

Production levels and activity of the cellulase fusion protein intransgenic maize made as in Example 5 or 6 is determined as follows.

Cellulase activity in transgenic maize is first assayed by standardmethods (Ghose. In Analytical Method B-304, rev. A, IUPAC Commission onBiotechnology. A short Report (1984)) based on the time course assay forhydrolysis of a pre-weighed sample of filter paper at pH 4.8-5.2 andtemperature of 50° C. While the filter paper assay is a standardsubstrate for cellulase activity, results using the filter paper assayare not particularly representative of the actual activity of thecellulase in plant materials containing cellulose, hemicellulose, andother sugars or sugar polymers. Therefore, a more accurate method fordetermining cellulase activity is used.

Plant material is ground and the ground material is suspended to aconcentration of up to about 5% in 0.05 M citrate buffer at pH 4.8 andincubated with shaking at 50° C. Over a 48 hour time period, samples areremoved at intervals of 0, 1, 3, 12, 24, and 48 hours. A minimal amountof sodium azide, about 0.05%, is added to the citrate buffer duringincubation to control microbial activity. For analysis by high pressureliquid chromatography (HPLC), the supernatant fraction of each sample isremoved, capped, and heated to inactivate the enzymes. The inactivatedsupernatant fraction is filtered through a syringe filter and analyzedby HPLC to measure the glucose, cellobiose, and xylose content of thesamples according to established methods (³⁹Dale et al., BiosourceTechnol. 56: 11-116 (1996)).

Cellulase activity is manifested by an increasing level of glucose,xylose and/or cellobiose levels in the supernatant fractions during the48 hour period. The control for the above assay is to treat samples fromnon-transgenic plants with varying amounts of commercially availablecellulase enzymes such as CYTOLASE 300 which is a cellulase fromGenencor, Inc. and NOVOZYME 188 which is a cellobiose from NovoLaboratories, Inc. to confirm that the ground plant material issusceptible to hydrolysis.

Example 8

Comparison of cellulase activity in transgenic maize prepared as inExample 5 or 6 treated to enhance cellulose accessibility.

Generally, cellulose and hemicellulose in plant material are not veryaccessible to hydrolytic enzymes such as cellulase. Therefore, it ispossible that even if the cellulase fusion protein is produced in thetransgenic plants of the present invention, its cellulase activity wouldnot be measurable. Therefore, to demonstrate accessibility, samples ofthe transgenic maize plants of the present invention are treated by theammonia fiber explosion process to increase cellulose and hemicelluloseaccessibility (³⁹Dale et al., Biosource technol. 56: 11-116 (1996)).Samples treated are analyzed as in Example 3.

In previous experiments with coastal Bermuda grass, the ammonia fiberexplosion process disrupted the plant cell walls sufficiently to permitover 80% extraction of plant protein, compared with less than 30%extraction under the same conditions prior to ammonia treatment (⁴⁰de laRosa et al., Appl. Biochem. Biotechnol. 45/46: 483-497 (1994). Theprocess increased the hydrolytic effectiveness of the added cellulasesby at least six-fold (³⁹Dale et al., Biosource Technol. 56: 11-116(1996)). Thus, it is expected that the ammonia fiber explosion processhelps release cellulase from the transgenic maize chloroplasts and willalso increase the access of the cellulase released to the cellulose inthe plant material.

Example 9

Production levels and activity of the ligninase fusion protein intransgenic maize made as in Example 5 or 6 can be determined as follows.

Maize leaves from the transgenic maize made as in Examples 5 or 6 areground using a pestle and mortar. Chloroplasts are isolated from leavesof transgenic plants by Ficoll (Pharmacia) gradient centrifugation andground as above.

The ground materials (leaves, grains, chloroplasts) are suspended in 50mM L-tartrate buffer (pH 4.5), mixed well by vortexing, and centrifugedfor 10 minutes at 14,000 rpm (16,000×g) at 4° C. and the supernatantfraction tested for lignin peroxidase (LIP) activity as described in⁴¹Tien et al., Meth. Enzymol. 161: 238-249 (1988). The LIP assaymeasures the production of veratraldehyde (as an increase in absorbanceat 310 nm) from veratryl alcohol (substrate) in the presence of hydrogenperoxide. Control assays are done on non-transgenic maize seeds tomeasure endogenous peroxidase activity. The assay is sensitive and isable to detect very low levels of lignin peroxidase activity, e.g.,conversion of 0.1 mmole substrate per minute per liter of test sample.

Soluble protein content is determined by the Bradford procedure(⁴²Bradford, Anal. Biochem. 72: 248-254 (1976)) using bovine serumalbumen (BSA) as the standard. LIP enzyme in the extracted fluid ispurified by Fast Protein liquid Chromatography (FPLC) analysis using theMono Q anion exchange system (Pharmacia) and a gradient of 0 to 1 MNa-acetate to elute the various isozymes CYadav et al., Appl. Environ.Microbiol. 61: 2560-2565 (1995); Reddy et al., FEMS Microbiol. Rev. 13:137-152 (1994)). The relative activity, yield, pH optimum, stability,and other characteristics of the LIP in the transgenic plant arecompared to that determined for the LIP isolated from the fungus.Furthermore, ground maize seeds or leaf extracts containing the LIP isused to treat various lignocellulosic feeds in small laboratory reactorsystems and the extent of delignification can be analyzed perestablished procedures (⁴⁴Van Soest et al., Assoc. Off. Anal. Chem. J.51: 780-785 (1968)).

Detection of ligninase mRNA is by isolating the mRNA from the transgenicplants as above, resolving the mRNA by denaturing RNA gelelectrophoresis, transferring the resolved mRNA to membranes, andprobing the membranes with ckg4 or ckg5 cDNA probes.

Western blots are performed to determine whether the LIP protein is inan active or inactive form. The total protein from the transgenic plantsis resolved by SDS-polyacrylamide gel electrophoresis and transferred tomembranes. The membranes are probed with antibodies to LIP H2 (ckg4) orLIP H10 (ckg5).

No one has produced cellulose hydrolysis enzymes and/or ligninases incereal cover crops. At present, cover crops are planted and destroyed.In the present invention, the transgenic cover crops are used forproduction of biofuels and biobased industrial material. As indicatedabove, cover crops are presently used to extend the season of activenutrient uptake and living soil cover. Therefore, they reduce nutrientlosses in water and sediment. They are also used to reduce soil erosion,reduce nitrogen leaching, and provide weed and pest suppression andincrease soil organic matter. In the present invention, all of the aboveadvantages are achieved. At present, herbicides are used to destroy thecover crops in spring before planting of other crops (cash crops). Inthis invention, herbicide application costs are unnecessary, thusproducing an additional benefit. The transgenic cover crops areharvested for biofuels and other biobased materials known in the art.

Two constructs were used, one with a hemicellulase (Xylanase or Xyl,such as Xyl1) (FIG. 16) and the other with a selectable maker (bar) gene(FIG. 17). In addition, two sets of ready made vectors were used thengenes are inserted for targeting of the enzymes in chloroplast, apoplast(cell wall areas), ER, vacuole or mitochondria. These two sets include(1) a set of vectors from Invitrogen called Gateway (FIGS. 18 and 19),and the other set called Impact Vectors from Europe. FIGS. 20 to 24 arediagrams of the constructs used to target the sub-cellular compartmentsof the plants.

While the present invention is described herein with reference toillustrated embodiments, it should be understood that the invention isnot limited hereto. Those having ordinary skill in the art and access tothe teachings herein will recognize additional modifications andembodiments within the scope thereof. Therefore, the present inventionis limited only by the Claims attached herein.

1.-21. (canceled)
 22. A method for providing transgenic plant materialfrom a cover crop with low amounts of lignin, the method comprising: (a)providing a transgenic monocot plant which is the low-lignin cover cropwhich includes (i) at least one DNA encoding a cellulase andhemicellulase which is operably linked to a nucleotide sequence encodinga signal peptide wherein the signal peptide directs the cellulase to anorganelle of the transgenic monocot plant and (ii) at least one DNAencoding a ligninase which is operably linked to a nucleotide sequenceencoding a signal peptide wherein the signal peptide directs theligninase to the organelle of the transgenic monocot plant; (b) growingthe transgenic monocot plant for a time sufficient for the transgenicmonocot plant to accumulate a sufficient amount of the cellulase, thehemicellulase, the ligninase in sub-cellular compartments of thetransgenic monocot plant; and (c) harvesting the transgenic monocotplant which has accumulated the cellulase, the hemicellulase, theligninase in the sub-cellular compartments of the transgenic monocotplant.
 23. The method of claim 22, wherein the transgenic monocot plantis selected from the group consisting of rice, wheat, barley, oat,triticale, and rye.
 24. The method of claim 22, wherein the sub-cellularcompartment of the transgenic monocot plant is selected from the groupconsisting of nucleus, microbody, endoplasmic reticulum, endosome,vacuole, mitochondria, chloroplast, and plastid.
 25. The method of claim22, wherein the sub-cellular compartment of the transgenic monocot plantis selected from the group consisting of chloroplast, apoplast,endoplasmic reticulum, mitochondria, and vacuole.
 26. The method ofclaim 22, wherein the DNA encoding the cellulase and hemicellulase andthe DNA encoding the ligninase are each operably linked to aleaf-specific promoter.
 27. The method of claim 22, wherein the DNAencoding the cellulase and hemicellulase and the DNA encoding theligninase are stably integrated into nuclear or plastid DNA of thetransgenic monocot plant.
 28. The method of claim 22, wherein: (i) theDNA encoding the cellulase is from an organism selected from the groupconsisting of Trichoderma reesei, Acidothermus cellulyticus,Streptococcus salivarius, Actinomyces naeslundi, and Thermomonosporafusca; (ii) the DNA encoding the hemicellulase is from Cochlioboluscarbonum; and (iii) the DNA encoding the ligninase is from Phanerochaetechrysosporium.
 29. The method of claim 22, wherein: (i) the DNA encodingthe cellulase is selected from the group consisting of an e1 genecomprising the nucleotide sequence set forth in SEQ ID NO:4 fromAcidothermus cellulyticus, a cbh1 gene comprising the nucleotidesequence set forth in SEQ ID NO:10 from Trichoderma reesei, a dextranasegene comprising the nucleotide sequence set forth in SEQ ID NO:8 fromStreptococcus salivarius, and a beta-glucosidase gene comprising thenucleotide sequence set forth in SEQ ID NO:6 from Actinomyces naeslundi;(ii) the DNA encoding the hemicellulase is a Xyl1 gene encoding axylanase; and (iii) the DNA encoding the ligninase is selected from thegroup consisting of a ckg4 gene comprising the nucleotide sequence setforth in SEQ ID NO:11, and a ckg5 gene comprising the nucleotidesequence set forth in SEQ ID NO:13.
 30. A method for providingtransgenic plant material from a cover crop with low amounts of lignin,the method comprising: (a) growing and harvesting a first cash crop on aplot of land; (b) providing a transgenic monocot plant which is thelow-lignin cover crop which includes at (i) least one DNA encoding acellulase and hemicellulase which is operably linked to a nucleotidesequence encoding a signal peptide wherein the signal peptide directsthe cellulase to an organelle of the transgenic monocot plant and (ii)optionally at least one DNA encoding a ligninase which is operablylinked to a nucleotide sequence encoding a signal peptide wherein thesignal peptide directs the ligninase to the organelle of the transgenicmonocot plant; (c) growing the transgenic monocot plant on the plot (i)during a winter season, (ii) subsequent to the harvesting of the firstcash crop from the plot, and (iii) for a time sufficient for thetransgenic monocot plant to accumulate a sufficient amount of thecellulase, the hemicellulase, and the ligninase if present insub-cellular compartments of the transgenic monocot plant; and (d)harvesting the transgenic monocot plant which has accumulated thecellulase, the hemicellulase, and the ligninase if present in thesub-cellular compartments of the transgenic monocot plant.
 31. Themethod of claim 30, wherein the first cash crop is corn.
 32. The methodof claim 30, wherein the first cash crop is soybean.
 33. The method ofclaim 30, wherein the transgenic monocot plant is selected from thegroup consisting of rice, wheat, barley, oat, triticale, and rye. 34.The method of claim 30, wherein the sub-cellular compartment of thetransgenic monocot plant is selected from the group consisting ofnucleus, microbody, endoplasmic reticulum, endosome, vacuole,mitochondria, chloroplast, and plastid.
 35. The method of claim 30,wherein the sub-cellular compartment of the transgenic monocot plant isselected from the group consisting of chloroplast, apoplast, endoplasmicreticulum, mitochondria, and vacuole.
 36. The method of claim 30,wherein the DNA encoding the cellulase and hemicellulase is stablyintegrated into nuclear or plastid DNA of the transgenic monocot plant.37. The method of claim 30, wherein the transgenic monocot plantincludes the at least one DNA encoding the ligninase.
 38. The method ofclaim 30, wherein: (i) the DNA encoding the cellulase is from anorganism selected from the group consisting of Trichoderma reesei,Acidothermus cellulyticus, Streptococcus salivarius, Actinomycesnaeslundi, and Thermomonospora fusca; and (ii) the DNA encoding thehemicellulase is from Cochliobolus carbonum.
 39. The method of claim 38,wherein the transgenic monocot plant includes the at least one DNAencoding the ligninase, and the DNA encoding the ligninase is fromPhanerochaete chlysosporium.
 40. The method of claim 30, wherein: (i)the DNA encoding the cellulase is selected from the group consisting ofan e1 gene comprising the nucleotide sequence set forth in SEQ ID NO:4from Acidothermus cellulyticus, a cbh1 gene comprising the nucleotidesequence set forth in SEQ ID NO:10 from Trichoderma reesei, a dextranasegene comprising the nucleotide sequence set forth in SEQ ID NO:8 fromStreptococcus salivarius, and a beta-glucosidase gene comprising thenucleotide sequence set forth in SEQ ID NO:6 from Actinomyces naeslundi;and (ii) the DNA encoding the hemicellulase is a Xyl1 gene encoding axylanase.
 41. The method of claim 40, wherein the transgenic monocotplant includes the at least one DNA encoding the ligninase, and the DNAencoding the ligninase is selected from the group consisting of a ckg4gene comprising the nucleotide sequence set forth in SEQ ID NO:11, and ackg5 gene comprising the nucleotide sequence set forth in SEQ ID NO:13.